Aryl receptor modulators and methods of making and using the same

ABSTRACT

The present invention is generally directed towards compounds capable of binding the aryl hydrocarbon receptor and modulating its activity, methods of treating inflammatory conditions such as Crohn&#39;s disease using such compounds, and pharmaceutical compositions comprising such compounds. Also provided are methods of increasing levels of IL-22 in a subject and/or decreasing levels of IFN-γ in a subject.

RELATED APPLICATIONS

This application is a continuation patent application of U.S. patentapplication Ser. No. 15/321,967, filed on Dec. 23, 2016, whichapplication is the U.S. national stage application under 35 U.S.C. § 371of International Application No. PCT/EP2015/064613, filed on Jun. 26,2015, which claims priority to and the benefit of U.S. ProvisionalApplication No. 62/017,959, filed on Jun. 27, 2014, and U.S. ProvisionalApplication No. 62/056,054, filed on Sep. 26, 2014, the entire contentsof each of which are incorporated by reference herein.

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Feb. 21, 2023, isnamed GIU-040C1_SL.xml and is 11,691 bytes in size.

BACKGROUND

Aryl hydrocarbon receptor (AhR), a basic helix-loop-helix protein, is amember of the Per-ARNT-Sim (PAS) superfamily of proteins.Physiologically, many of these proteins act by sensing molecules andstimuli from the cellular/tissue microenvironment, thereby initiatingsignaling cascades necessary to elicit appropriate cellular responses.

In its inactive state, AhR resides in the cytosol bound to severalco-chaperones, but after activation, it migrates into the nucleus andbinds its dimerization partner, ARNT (another bHLH-PAS protein), thusinitiating the transcription of a variety of genes with promoterscontaining a dioxin (DRE) or xenobiotic consensus sequence (XME).Pioneering studies in AhR-deficient mice have emphasized the role of AhRin the development and functions of various organs. More recent studieshave shown that AhR controls specific immune responses (see, forexample, Stockinger et al., Semin. Immunol. 2011, 23, 99-105).

AhR is highly expressed by T cells and controls Th1/Th2/Th17cell-associated immunity. In humans and in mouse models of inflammatorybowel disease, activation of AhR led to a diminished expression of IFN-7and T-bet, the main transcription factor that drives the Th1polarization. The basic mechanism by which AhR inhibits IFN-γ productionremains to be ascertained, however, it has been demonstrated that AhRsignaling enhances Aiolos, a member of the Ikaros family, thatnegatively regulates IFN-γ production and colitis in mice. AhRactivation in lymphoid cells can also regulate production ofinterleukin-22 (IL-22), a cytokine that can exert protective effects invarious organs and time-course studies showed that suppression of IFN-γand T-bet preceded IL-22 induction.

In light of the potential for selective AhR modulators to affectimmunity and inflammation and thus treat a variety of inflammatoryconditions, there exists a need for potent and selective compounds thatmodulate AhR activity.

SUMMARY

The present disclosure, in an embodiment, provides potent and selectiveAhR-binding compounds. In one aspect, the present disclosure provides acompound according to Formula I:

wherein the variables are as defined below.

In one aspect, the present disclosure relates to a compound according toFormula I-A:

wherein the variables are as defined below.

In one aspect, the present disclosure relates to a compound according toFormula II:

wherein the variables are as defined below.

In one aspect, the present disclosure relates to a compound according toFormula III:

wherein the variables are as defined below.

In one aspect, the present disclosure relates to a compound according toFormula IV:

wherein the variables are as defined below.

The present disclosure also provides methods of treating an inflammatorydisease or condition, comprising administering to a subject in needthereof a therapeutically effective amount of a compound describedabove. In certain embodiments, the inflammatory disease or condition isselected from the group consisting of inflammatory bowel disease,cartilage inflammation, bone degradation, ulcerative colitis, psoriasis,arthritis, psoriatic arthritis, rheumatoid arthritis, juvenilearthritis, juvenile rheumatoid arthritis, autoimmune hepatitis, Crohn'sdisease, lupus erythematosus, multiple sclerosis, Alzheimer's disease,dermatitis, atopic dermatitis, acne, Type I diabetes mellitus, Raynaud'sphenomenon, Graves' disease, and Addison's disease.

In certain embodiments, the inflammatory disease or condition isselected from the group consisting of Crohn's disease, ulcerativecolitis, collagenous colitis, lymphocytic colitis, diversion colitis,Behçet's disease, idiopathic inflammatory bowel disease, irritable bowelsyndrome, regional enteritis, spastic colon, microscopic colitis,Crohn's colitis, perianal disease, indeterminate colitis, lymphocyticgastritis, and eosinophilic enteritis.

In certain embodiments, the inflammatory disease or condition is Crohn'sdisease. In certain embodiments, the Crohn's disease is selected fromthe group consisting of ileocolitis, ileitis, gastroduodenal Crohn'sdisease, jejunoileitis, and granulomatous ileocolitis. In certainembodiments, the Crohn's disease includes intestinal fibrosis. Incertain embodiments, the Crohn's disease is fibrostenotic Crohn'sdisease.

In another aspect, the present disclosure provides a method ofpreventing, treating, or reducing fibrostenosis or intestinal fibrosisin a subject, comprising administering to a subject in need thereof atherapeutically effective amount of a compound disclosed herein. Incertain embodiments, the fibrostenosis or intestinal fibrosis isassociated with Crohn's disease.

In another aspect, the present invention provides a compound asdescribed above for use as a medicament. The present invention furtherprovides a compound as described above for use in a method of treatingan inflammatory disease or condition. The method comprises administeringto a subject in need thereof a therapeutically effective amount of saidcompound. In certain embodiments, the inflammatory disease or conditionis selected from the group consisting of inflammatory bowel disease,cartilage inflammation, bone degradation, ulcerative colitis, psoriasis,arthritis, psoriatic arthritis, rheumatoid arthritis, juvenilearthritis, juvenile rheumatoid arthritis, autoimmune hepatitis, Crohn'sdisease, lupus erythematosus, multiple sclerosis, Alzheimer's disease,dermatitis, atopic dermatitis, acne, Type I diabetes mellitus, Raynaud'sphenomenon, Graves' disease, and Addison's disease. In certainembodiments, the inflammatory disease or condition is selected from thegroup consisting of Crohn's disease, ulcerative colitis, collagenouscolitis, lymphocytic colitis, diversion colitis, Behçet's disease,idiopathic inflammatory bowel disease, irritable bowel syndrome,regional enteritis, spastic colon, microscopic colitis, Crohn's colitis,perianal disease, indeterminate colitis, lymphocytic gastritis, andeosinophilic enteritis. In certain embodiments, the inflammatory diseaseor condition is Crohn's disease. In certain embodiments, the Crohn'sdisease is selected from the group consisting of ileocolitis, ileitis,gastroduodenal Crohn's disease, jejunoileitis, and granulomatousileocolitis. In certain embodiments, the Crohn's disease includesintestinal fibrosis. In certain embodiments, the Crohn's disease isfibrostenotic Crohn's disease.

In yet another aspect, the present invention provides the compound asdescribed above for use in a method of preventing, treating or reducingfibrostenosis or intestinal fibrosis in a subject. The method comprisesadministering to a subject in need thereof a therapeutically effectiveamount of said compound. In certain embodiments, the fibrostenosis orintestinal fibrosis is associated with Crohn's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a series of 1-aryl-1,2,3,4-tetrahydro-β-carbolinederivatives.

FIG. 2 shows modifications made to the1-aryl-1,2,3,4-tetrahydro-β-carboline scaffold to study the SAR andprovide improved properties.

FIG. 3 shows modifications made to the1-aryl-1,2,3,4-tetrahydro-β-carboline scaffold to study the SAR andprovide improved properties.

FIG. 4 shows modifications made to the1-aryl-1,2,3,4-tetrahydro-β-carboline scaffold to study the SAR andprovide improved properties.

FIG. 5 shows modifications made to the Leflunomide scaffold to study theSAR and provide improved properties.

FIG. 6 shows the chemical structures of various compounds evaluated forADMET and other properties in the present disclosure.

FIG. 7 shows the chemical structures of various compounds evaluated forADMET and other properties in the present disclosure.

FIG. 8 shows the chemical structures of various compounds evaluated forADMET and other properties in the present disclosure.

FIG. 9 shows the chemical structures of various compounds evaluated forADMET and other properties in the present disclosure.

FIG. 10 shows calculated bioavailability data for compounds of thepresent disclosure. All ADME data were calculated using ADME Suitev4.95.3. Human oral bioavailability (% F) Human oral bioavailability (%F) is the compound fraction that reaches systematic circulation afteroral administration. In order to be bioavailable, a drug must complywith the following requirements: dissolve in the stomach or intestineunder variable pH; withstand acid hydrolysis at pH<2; permeate throughintestinal membrane by passive or active transport; withstand P-gpefflux in concert with metabolic enzymes in intestine; and withstandfirst pass metabolism in liver.

FIG. 11A and FIG. 11B show calculated aqueous solubility (log Sw) ofvarious compounds of the present disclosure. Solubility (log Sw) of acompound in pure water at 25° C. and at various physiologicallyimportant pH values was predicted.

FIG. 12A and FIG. 12B show calculated pH dependent solubility forvarious compounds of the present disclosure.

FIG. 13 shows calculated human intestinal passive absorption for variouscompounds of the present disclosure. The human intestinal permeabilitywas assessed through estimation of maximum intestinal passive absorptionof a compound taking into account the transcellular and paracellularroutes of permeability. Absorption related properties such as jejunumand Caco-2 permeabilities, and absorption rates (ka) values were alsocalculated using lipophilicity (log P) and ionization (pKa) constants.

FIG. 14 shows calculated active transport across the intestinal barrierfor various compounds of the present disclosure. The calculatedtransport included estimation of PepT1 (oligopeptide transporter) andASBT (bile acid transporter) substrate prediction.

FIG. 15 shows calculated P-glycoprotein specificity for variouscompounds of the present disclosure. The algorithm identifies P-gpsubstrates and/or inhibitors. Substrates are compounds that aretransported (effluxed) by P-gp. Inhibitors are compounds that block P-gptransport of the standard substrates (calcein-AM and others).

FIG. 16 shows calculated tissue distribution of various compound of thepresent disclosure. The software predicts the extent of plasma proteinbinding to obtain the percentage of compound that circulates in free,pharmacologically active form, and calculates the apparent volume ofdistribution for an estimation of the distribution of compounds betweenplasma and body tissue.

FIG. 17 shows calculated interaction of various compounds of the presentdisclosure with cytochrome P450s. The software calculates how compoundswill interact with the five cytochrome P450 (CYP) isoforms: 3A4, 2D6,2C9, 2C19, and 1A2, that are responsible for the majority of metabolicreactions.

FIG. 18 shows data calculated with Tox Suite v 2.95 expressing thelikelihood of genotoxicity resulting from various compounds of thepresent disclosure. Genotoxicity=Probability of positive Ames test. TheAmes test is one of the most popular tests for assessing mutagenicproperties of compounds. It is a short term bacterial reverse mutationtest. This test is performed on various S. typhimurium and E. colibacteria strains. The Ames test is used worldwide as an initial screento determine mutagenic properties of NCEs in the drug and chemicalindustry.

FIG. 19 shows data expressing the calculated likelihood that variouscompounds of the present disclosure will inhibit hERG. Studies of hERGpotassium ion channel inhibition constitute an emerging field inpharmacological safety research. Interactions of drugs with the hERGchannel may lead to long QT syndrome, manifesting as characteristic‘Torsades de Pointes’ arrhythmia, leading to occasional fatality causedby ventricular fibrillation. In recent years several promising drugshave been withdrawn from the market due to a number of sudden cardiacdeath occasions triggered by hERG channel inhibition. Thus, earlyidentification of leads possessing potential safety issues is of extremeimportance to prevent costly failures of R&D projects.

FIG. 20 shows the calculated probability of health effects for variouscompounds of the present disclosure. Predictions on health effects arebased on long-term toxicity studies with adverse effects reported onparticular organs or organ systems.

FIG. 21 shows calculated LD₅₀ values for various compounds of thepresent disclosure. LD₅₀ values can be viewed as an indication of the“cumulative potential” to cause various acute effects and death ofanimals and are the most widely used measure of the “acute systemictoxicity” of the chemical.

FIG. 22 shows the effects, at a final concentration of 200 nM, ofvarious compounds of the present disclosure on IL-22 and IFN-γ levels.

FIG. 23 shows the effects of various compounds of the present disclosureon IL-22 and IFN-γ levels, as well as the chemical structure of knownAhR binder Ficz.

FIG. 24 shows the effects of various compounds of the present disclosureon IL-22 and IFN-γ levels. For each entry, the first number refers tothe compound number and the second number refers to the concentration innM; thus, “02 100” refers to administration of compound 2 at aconcentration of 100 nM, “04 200” to administration of compound 4 at aconcentration of 200 nM, etc.

FIG. 25A shows evaluation of AhR transcripts in fibroblasts isolatedfrom gut mucosa of 6 normal patients (control group; CTR), 7 patientswith ulcerative colitis (UC), and 7 with Crohn's disease (CD) by realtime PCR and normalized to β-actin. FIG. 25B shows flow cytometryanalysis of AhR in fibroblasts isolated from 5 normal patients (CTR), 5patients with UC, and 5 patients with CD. Right insets showrepresentative histograms of AhR-expressing fibroblasts isolated fromone CTR, one patient with UC, and one patient with CD. Staining with anisotype control IgG is also shown. Data indicate mean+/−SD of allexperiments. Taken together, these data demonstrate that intestinalfibroblasts constitutively express AhR.

FIG. 26A shows the results of stimulating Crohn's disease (CD)intestinal fibroblasts with TGF-β (1 ng/mL) in the presence or absenceof Ficz (100, 200, or 400 nM). FIG. 26B shows the results of stimulatingCD intestinal fibroblasts with TNF-α (15 ng/mL) in the presence orabsence of Ficz (100, 200, or 400 nM). Col1A1, Col3A1, and α-SMA wereexamined by real-time PCR after 24 hours. Data indicate mean+/−SD of 3experiments. Taken together, these data demonstrate that AhR activationinhibits fibroblast collagen expression induced by pro-fibroticcytokines.

FIG. 27A shows the results of stimulating Crohn's disease (CD)intestinal fibroblasts with TGF-β (1 ng/mL) in the presence or absenceof CH223191 (a-AhR, final concentration 10 μM). FIG. 27B shows theresults of stimulating CD intestinal fibroblasts with TNF-α (15 ng/mL)in the presence or absence of CH223191 (a-AhR, final concentration 10PM). Col1A1, Col3A1, and α-SMA were analyzed by real-time PCR after 24hours. Data indicate mean+/−SD of 3 experiments.

FIGS. 28A-28D show total collagen analysis of supernatants of Crohn'sdisease (CD) fibroblasts stimulated with TGF-β (1 ng/mL) (FIGS. 28A andC) or TNF-α (15 ng/mL) (FIGS. 28B and D) in the presence or absence ofFicz (FIGS. 28A and B) or CH223191 (a-AhR, final concentration 10 μM)(FIGS. 28C and D) for 48 hours. Data indicate mean+/−SD of 3experiments. Taken together, these data show that AhR controls collagensecretion.

FIG. 29A shows percentages of CD intestinal fibroblasts expressingp-p38+, p-ERK1/2+, p-NF-kBp65+, or p-Smad2/3+ that were eitherunstimulated (Unst) or stimulated with TGF-β (1 ng/mL) or TNF-α (15ng/mL) in the presence or absence of Ficz (final concentration 200 nM)or CH223191 (a-AhR, final concentration 10 μM) for 24 h. Assessment ofp-P38 (pT180/pY182), p-ERK1/2(pT202/pY204)(pT184/pY186), p-NF-kBp65, andp-Smad2/3 was accomplished by flow cytometry. Numbers indicate thepercentages of p-p38+, p-ERK1/2+, p-NF-kBp65+, or p-Smad2/3+ cells inthe designated gates. Isotype control stain is also indicated. One of 3representative experiments in which cells of 3 patients were used isshown. FIG. 29B shows percentages of p-p38+, p-ERK1/2+, p-NF-kBp65+, orp-Smad2/3+ fibroblasts isolated from 3 CD patients stimulated eitherwith TGF-β (1 ng/mL) or TNF-α (15 ng/mL) in the presence or absence ofFicz (final concentration 200 nM) or CH223191 (a-AhR, finalconcentration 10 μM) for 24 h. Data indicate the mean±SD of 3experiments. * p<0.04 vs. unstimulated; ** p<0.001 vs. unstimulated;#p<0.03 vs. TGF-β; +p<0.02 vs. TNF-α. Taken together, these datademonstrate that AhR activation leads to inactivation of p38 and ERK1/2in Crohn's disease (CD) fibroblasts.

FIG. 30A shows a schematic view of TNBS-induced intestinal fibrosismodel. Balb/c mice were given weekly TNBS treatments and Ficz orCH223191 (a-AhR) were administered starting after the fifth TNBSadministration. FIG. 30B shows representative colon cross-sections ofcontrol (CTR) mice and TNBS-treated mice receiving Ficz or CH223191(a-AhR) stained with Masson's trichrome. Percentages of animalsharboring mild, moderate, and severe fibrosis are also indicated. FIG.30C shows relative RNA expression data for Col1A2 in colonic samplestaken from CTR mice and TNBS-treated mice injected with Ficz or CH223191(a-AhR) analyzed by real-time PCR. FIG. 30D shows total collagen contentdata (μg/mg) analyzed by colorimetric assay. Data indicate mean+/−SD of3 separate experiments (n=12 mice total per group). Taken together,these data demonstrate that Ficz-treated mice are largely protected fromTNBS-induced intestinal fibrosis.

DETAILED DESCRIPTION

In one aspect, the present disclosure provides a compound according toFormula I:

or a pharmaceutically acceptable salt thereof, wherein:

A is

Ar is represented by

G¹ is CR⁴ or N;

G², G³, and G⁴ are each independently CR⁴ ₂ or NR¹;

X¹ is independently for each occurrence H, halogen, —OH, —CN, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₆ cycloalkyl, C₁₋₆ alkoxy,—O-phenyl, —N(R¹)₂, —NO₂, —C₁₋₆ alkylene-N(R¹)₂, —C(O)N(R³)₂, —CO₂R³,—C(O)R³, —SR³, —SO₂R³, —SO₃R³, or —SO₂N(R³)₂;

X² is independently for each occurrence H, halogen, —OH, —CN, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₆ cycloalkyl, C₁₋₆ alkoxy,—O-phenyl, —N(R¹)₂, —NO₂, —C₁₋₆ alkylene-N(R¹)₂, —C(O)N(R³)₂, —CO₂R³,—SR³, —SO₂R³, —SO₃R³, or —SO₂N(R³)₂;

R¹ is independently for each occurrence H or C₁₋₆ alkyl;

R² is independently for each occurrence H or C₁₋₆ alkyl;

R³ is independently for each occurrence selected from the groupconsisting of H, C₁₋₆ alkyl, phenyl, or heteroaryl;

R⁴ is independently for each occurrence selected from the groupconsisting of H, C₁₋₆ alkyl, and halogen;

n is independently for each occurrence 0, 1, 2, or 3; and

m is independently for each occurrence 0, 1, 2, 3, or 4;

wherein each instance of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₆cycloalkyl, C₁₋₆ alkoxy, or —O-phenyl may be optionally substituted with1, 2, or 3 substituents each independently selected from the groupconsisting of halogen, —OH, —CN, —NR′R″—C(O)N(R′R″), and —C(O)R′(wherein R′ and R″ are each independently selected from H, methyl,ethyl, propyl or butyl, or R′ and R″ taken together form a 4-6 memberedheterocycle);

wherein when A is

G¹ is CH, G³, and G⁴ are CH₂, and G² is NR¹, X² is not H (i.e., is notpresent, n is 0).

In certain embodiments, G¹ is N.

In certain embodiments, G² is NH.

In certain embodiments, G¹ is CH, G² is NH, and G³ and G⁴ are CH₂.

In certain embodiments, G¹ is N and G², G³, and G⁴ are CH₂.

In certain embodiments, G¹ is N, G² and G³ are CH₂, and G⁴ is NH.

In certain embodiments, G¹ is N, G² and G⁴ are CH₂, and G³ is NH.

In certain embodiments, G¹ is CH, G² and G⁴ are NH, and G³ is CH₂.

In certain embodiments, Ar is

and m is 1 or 2.

In certain embodiments, Ar is

In certain embodiments, X¹ is H, halogen, —CN, or —OMe.

In certain embodiments, X¹ is —OMe.

In certain embodiments, X² is selected from the group consisting of H,halogen, —OH, —CN, C₁₋₆ alkoxy, and CF₃.

In certain embodiments, X² is CF₃ and n is 1.

In certain embodiments, the compound is selected from the groupconsisting of

In one aspect, the present disclosure relates to a compound according toFormula I-A:

or a pharmaceutically acceptable salt thereof, wherein:

X¹ is if present independently for each occurrence C₁₋₆ alkyl, C₁₋₆alkoxy, —O—C₁₋₆ alkylene-phenyl, C₁₋₆ acyl, —CO₂R³, —NO₂, —OH, or—N(R¹)₂; or two instances of X¹ taken together may be —O(CH₂)₂O—;

X² is selected from the group consisting of H, C₁₋₆ alkyl, C₁₋₆ alkoxy,—O—C₁₋₆ alkylene-phenyl, and —OH;

R¹ is independently for each occurrence H or C₁₋₆ alkyl;

R³ is independently for each occurrence H, C₁₋₆ alkyl, phenyl, orheteroaryl;

R⁵ is selected from the group consisting of H, C₁₋₆ alkyl, C₁₋₆ acyl,and —CO₂R³; and

n is 0, 1, 2, or 3;

wherein each instance of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₆cycloalkyl, C₁₋₆ alkoxy, or —O-phenyl may be optionally substituted with1, 2, or 3 substituents each independently selected from the groupconsisting of halogen, —OH, —CN, —NR′R″—C(O)N(R′R″), and —C(O)R′(wherein R′ and R″ are each independently selected from H, methyl,ethyl, propyl or butyl, or R′ and R″ taken together form a 4-6 memberedheterocycle).

In certain embodiments, X² is selected from the group consisting of H,—OMe, ethyl, —OH, and —OCH₂Ph.

In certain embodiments, R⁵ is selected from the group consisting of H,acetyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl,isopentyl, and —CO₂CH₂Ph.

In certain embodiments, X¹ is independently for each occurrence H, —OMe,—NO₂, —CO₂Me, methyl, —OC(O)Me, —OCH₂Ph, —OH, —NH₂, or tert-butyl; ortwo instances of X¹ taken together may be —O(CH₂)₂O—.

In certain embodiments, n is 3.

In one aspect, the present disclosure relates to a compound according toFormula II:

or a pharmaceutically acceptable salt thereof, wherein:

B is

C is

Ar is represented by

X¹ is independently for each occurrence, if present, selected fromhalogen, —OH, —CN, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₆cycloalkyl, C₁₋₆ alkoxy, —O-phenyl, —N(R¹)₂, —NO₂, —C₁₋₆alkylene-N(R¹)₂, —C(O)N(R³)₂, —CO₂R³, —C(O)R³, —SR³, —SO₂R³, —SO₃R³, or—SO₂N(R³)₂;

X² is independently for each occurrence if present, selected from,halogen, —OH, —CN, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₆cycloalkyl, C₁₋₆ alkoxy, —O-phenyl, —N(R¹)₂, —NO₂, —C₁₋₆alkylene-N(R¹)₂, —C(O)N(R³)₂, —CO₂R³, —SR³, —SO₂R³, —SO₃R³, or—SO₂N(R³)₂;

R¹ is independently for each occurrence H or C₁₋₆ alkyl;

R³ is independently for each occurrence H, C₁₋₆ alkyl, phenyl, orheteroaryl;

R⁴ is selected from the group consisting of H, C₁₋₆ alkyl, C₂₋₆ alkenyl,C₂₋₆ alkynyl, —N(R¹)₂, —C₁₋₆ alkylene-N(R¹)₂, —C(O)N(R³)₂, and —CO₂R³;

n is independently for each occurrence 0, 1, 2, or 3;

m is independently for each occurrence 0, 1, 2, 3, or 4;

wherein each instance of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₆cycloalkyl, C₁₋₆ alkoxy, or —O-phenyl may be optionally substituted with1, 2, or 3 substituents each independently selected from the groupconsisting of halogen, —OH, —CN, —NR′R″—C(O)N(R′R″), and —C(O)R′(wherein R′ and R″ are each independently selected from H, methyl,ethyl, propyl or butyl, or R′ and R″ taken together form a 4-6 memberedheterocycle);

X³ is N or CR⁴;

X⁴ is NR¹, O, or S; and

Y is a bond, C₁₋₆ alkylene or —N(R¹)—;

wherein when B is

and X⁴ is S, X² is not H (i.e., is not present).

In certain embodiments of Formula II Y is CH₂ or NH.

In certain embodiments, R⁴ is selected from the group consisting of C₂₋₆alkenyl, C₂₋₆ alkynyl, —C₁₋₆ alkylene-N(R¹)₂, and —C(O)N(R³)₂.

In certain embodiments, R⁴ is allyl or

In certain embodiments, Ar is

and m is 1 or 2.

In certain embodiments, Ar is

In certain embodiments, X¹ is H (i.e., n is 0), halogen, —CN, or —OMe.

In certain embodiments, A, B and L taken together are compoundsrepresented by:

In certain embodiments, X² is selected from the group consisting of H,halogen, —OH, —CN, C₁₋₆ alkoxy, and CF₃.

In certain embodiments, X² is CF₃ and n is 1.

In one aspect, the present disclosure relates to a compound according toFormula III:

or a pharmaceutically acceptable salt thereof, wherein:

D is

Ar is represented by

X¹ may be present, and may be selected independently for each occurrencefrom, halogen, —OH, —CN, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₆cycloalkyl, C₁₋₆ alkoxy, —O-phenyl, —N(R¹)₂, —NO₂, —C₁₋₆alkylene-N(R¹)₂, —C(O)N(R³)₂, —CO₂R³, —C(O)R³, —SR³, —SO₂R³, —SO₃R³, or—SO₂N(R³)₂;

X² may be present, and may be selected independently for each occurrencefrom, halogen, —OH, —CN, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₆cycloalkyl, C₁₋₆ alkoxy, —O-phenyl, —N(R¹)₂, —NO₂, —C₁₋₆alkylene-N(R¹)₂, —C(O)N(R³)₂, —CO₂R³, —SR³, —SO₂R³, —SO₃R³, or—SO₂N(R³)₂;

R¹ is independently for each occurrence H or C₁₋₆ alkyl;

R³ is independently for each occurrence H, C₁₋₆ alkyl, phenyl, orheteroaryl;

n is independently for each occurrence 0, 1, 2, or 3; and

m is independently for each occurrence 0, 1, 2, 3, or 4;

wherein each instance of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₆cycloalkyl, C₁₋₆ alkoxy, or —O-phenyl may be optionally substituted with1, 2, or 3 substituents each independently selected from the groupconsisting of halogen, —OH, —CN, —NR′R″—C(O)N(R′R″), and —C(O)R′(wherein R′ and R″ are each independently selected from H, methyl,ethyl, propyl or butyl, or R′ and R″ taken together form a 4-6 memberedheterocycle);

wherein when D is

X² is not H (i.e., X² is not present and n is 0).

In certain embodiments, Ar is

and m is 1 or 2.

In certain embodiments, Ar is

In certain embodiments, X¹ is H, halogen, —CN, or —OMe.

In certain embodiments, X¹ is —OMe.

In certain embodiments, X² is selected from the group consisting of H,halogen, —OH, —CN, C₁₋₆ alkoxy, and CF₃.

In certain embodiments, X² is CF₃ and n is 1.

In one aspect, the present disclosure relates to a compound according toFormula IV:

or a pharmaceutically acceptable salt thereof, wherein:

E is a ring selected from the group consisting of heterocyclyl,5-membered heteroaromatic, 6-membered heteroaromatic, aromatic, and C₃₋₆cycloalkyl, wherein the ring is optionally substituted with 1, 2, or 3X²;

Ar is represented by

X¹ is if present, selected independently for each occurrence from thegroup consisting of H, halogen, —OH, —CN, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₆ cycloalkyl, C₁₋₆ alkoxy, —O-phenyl, —N(R¹)₂, —NO₂, —C₁₋₆alkylene-N(R¹)₂, —C(O)N(R³)₂, —CO₂R³, —C(O)R³, —SR³, —SO₂R³, —SO₃R³, or—SO₂N(R³)₂;

X² is independently for each occurrence selected from the groupconsisting of halogen, —OH, —CN, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,C₃₋₆ cycloalkyl, C₁₋₆ alkoxy, —O-phenyl, —N(R¹)₂, —NO₂, —C₁₋₆alkylene-N(R¹)₂, —C(O)N(R³)₂, —CO₂R³, —SR³, —SO₂R³, —SO₃R³, or—SO₂N(R³)₂; Y is selected from the group consisting of a bond, NR′ orC₁₋₆alkylene (e.g., a bond or C₁₋₂alkylene);

R¹ is independently for each occurrence H or C₁₋₆ alkyl;

R³ is independently for each occurrence selected from the groupconsisting of H, C₁₋₆ alkyl, phenyl, or heteroaryl; and

m is independently for each occurrence 0, 1, 2, 3, or 4;

wherein each instance of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₆cycloalkyl, C₁₋₆ alkoxy, or —O-phenyl may be optionally substituted with1, 2, or 3 substituents each independently selected from the groupconsisting of halogen, —OH, —CN, —NR′R″—C(O)N(R′R″), and —C(O)R′(wherein R′ and R″ are each independently selected from H, methyl,ethyl, propyl or butyl, or R′ and R″ taken together form a 4-6 memberedheterocycle).

In certain embodiments, of Formula IV, Y is a bond, CH₂ or NH; (forexample Y may be a bond).

In certain embodiments, Ar is

and m is 1 or 2.

In certain embodiments, Ar is

In certain embodiments, X¹ is H, halogen, —CN, or —OMe.

In certain embodiments, X¹ is —OMe.

In certain embodiments, X² is selected from the group consisting of H,halogen, —OH, —CN, C₁₋₆ alkoxy, and CF₃.

In certain embodiments, X² is CF₃.

In one aspect, the present disclosure relates to a pharmaceuticalcomposition, comprising a compound described above; and apharmaceutically acceptable carrier.

Definitions

The term “saturated” as used herein means the compound or group somodified has no carbon-carbon double and no carbon-carbon triple bonds,except as noted below. The term does not preclude carbon-heteroatommultiple bonds, for example a carbon oxygen double bond or a carbonnitrogen double bond. Moreover, it does not preclude a carbon-carbondouble bond that may occur as part of keto-enol tautomerism orimine/enamine tautonerisn.

The term “alkyl” refers to a monovalent saturated aliphatic group with acarbon atom as the point of attachment, a linear or branched, cyclic oracyclic structure, and no atoms other than carbon and hydrogen. Thus, asused herein cycloalkyl is a subset of alkyl. The groups —CH₃ (Me),—CH₂CH₃ (Et), —CH₂CH₂CH₃ (n-Pr), —CH(CH₃)₂ (i-Pr), —CH(CH₂)₂(cyclopropyl), —CH₂CH₂CH₂CH₃ (n-Bu), —CH(CH₃)CH₂CH₃ (sec-butyl),—CH₂CH(CH₃)₂ (iso-butyl), —C(CH₃)₃ (tert-butyl), —CH₂C(CH₃)₃(neopentyl), cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexylmethylare non-limiting examples of alkyl groups. The term “alkylene” refers toa divalent saturated aliphatic group, with one or two saturated carbonatom(s) as the point(s) of attachment, a linear or branched, cyclo,cyclic or acyclic structure, no carbon-carbon double or triple bonds,and no atoms other than carbon and hydrogen. The groups, —CH₂—(methylene), —CH₂CH₂—, —CH₂C(CH₃)₂CH₂—, —CH₂CH₂CH₂—, and

are non-limiting examples of alkylene groups.

The term “alkenyl” refers to a monovalent unsaturated aliphatic groupwith a carbon atom as the point of attachment, a linear or branched,cyclic or acyclic structure, at least one nonaromatic carbon-carbondouble bond, no carbon-carbon triple bonds, and no atoms other thancarbon and hydrogen. Non-limiting examples of alkenyl groups include:—CH═CH₂ (vinyl), —CH═CHCH₃, —CH═CHCH₂CH₃, —CH₂CH═CH₂ (allyl),—CH₂CH═CHCH₃, and —CH═CH—C₆H₅.

The term “alkynyl” refers to an monovalent unsaturated aliphatic groupwith a carbon atom as the point of attachment, a linear or branched,cyclo, cyclic or acyclic structure, at least one carbon-carbon triplebond, and no atoms other than carbon and hydrogen. As used herein, theterm alkynyl does not preclude the presence of one or more non-aromaticcarbon-carbon double bonds. The groups —C≡CH, —C≡CCH₃, and —CH₂C≡CCH₃,are non-limiting examples of alkynyl groups.

The term “aryl” refers to a monovalent group with an aromatic carbonatom as the point of attachment, said carbon atom forming part of one ormore six-membered aromatic ring structure(s) wherein the ring atoms areall carbon, and wherein the monovalent group consists of no atoms otherthan carbon and hydrogen. Non-limiting examples of aryl groups includephenyl (Ph), methylphenyl, (dimethyl)phenyl, −C₆H₄—CH₂CH₃ (ethylphenyl),—C₆H₄—CH₂CH₂CH₃ (propylphenyl), —C₆H₄—CH(CH₃)₂, —C₆H₄—CH(CH₂)₂,—C₆H₃(CH₃)CH₂CH₃ (methylethylphenyl), —C₆H₄—CH═CH₂ (vinylphenyl),—C₆H₄—CH═CHCH₃, —C₆H₄C≡CH, —C₆H₄C≡CCH₃, naphthyl, and the monovalentgroup derived from biphenyl.

The term “heteroaryl” refers to a monovalent aromatic group with anaromatic carbon atom or nitrogen atom as the point of attachment, saidcarbon atom or nitrogen atom forming part of one or more aromatic ringstructures wherein at least one of the ring atoms is nitrogen, oxygen orsulfur, and wherein the heteroaryl group consists of no atoms other thancarbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromaticsulfur. As used herein, the term does not preclude the presence of oneor more alkyl, aryl, and/or aralkyl groups (carbon number limitationpermitting) attached to the aromatic ring or aromatic ring system. Ifmore than one ring is present, the rings may be fused or unfused.Non-limiting examples of heteroaryl groups include furanyl, imidazolyl,indolyl, indazolyl, isoxazolyl, methylpyridinyl, oxazolyl,phenylpyridinyl, pyridinyl, pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl,quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, andtriazolyl.

The term “heterocyclyl” refers to a monovalent non-aromatic group with acarbon atom or nitrogen atom as the point of attachment, said carbonatom or nitrogen atom forming part of one or more non-aromatic ringstructures wherein at least one of the ring atoms is nitrogen, oxygen orsulfur, and wherein the heterocyclyl group consists of no atoms otherthan carbon, hydrogen, nitrogen, oxygen and sulfur. As used herein, theterm does not preclude the presence of one or more alkyl groups (carbonnumber limitation permitting) attached to the ring or ring system. Ifmore than one ring is present, the rings may be fused or unfused.Non-limiting examples of heterocyclyl groups include aziridinyl,pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl,tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, andpyranyl.

The term “acyl” refers to the group —C(O)R, in which R is a hydrogen,alkyl, aryl, aralkyl or heteroaryl, as those terms are defined above.The groups —CHO, —C(O)CH₃ (acetyl, Ac), —C(O)CH₂CH₃, C(O)CH₂CH₃,—C(O)CH(C₃)₂, —C(O)CH(CH₂)₂, —C(O)C₆H₅, —C(O)C₆H₄—CH₃, —C(O)CH₂C₆H₅,—C(O)(imidazolyl) are non-limiting examples of acyl groups. A “thioacyl”is defined in an analogous manner, except that the oxygen atom of thegroup —C(O)R has been replaced with a sulfur atom.

The term “alkoxy” refers to the group —OR, in which R is an alkyl, asthat term is defined above. Non-limiting examples of alkoxy groupsinclude: —OCH₃, —OCH₂CH₃, —OCH₂CH₂CH₃, —OCH(CH₃)₂, —OCH(CH₂)₂,—O-cyclopentyl, and —O-cyclohexyl.

The definition of “pharmaceutically acceptable” is meant to encompassany carrier, salt form, or other agent, which does not interfere witheffectiveness of the biological activity of the active ingredient andthat is not toxic to the host to which it is administered. As usedherein, “pharmaceutically acceptable carrier” means buffers, carriers,diluents, and excipients suitable for use in contact with the tissues ofhuman beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio. The carrier(s) should be “acceptable” inthe sense of being compatible with the other ingredients of theformulations and not deleterious to the recipient. Pharmaceuticallyacceptable carriers include buffers, solvents, dispersion media,coatings, isotonic and absorption delaying agents, and the like, thatare compatible with pharmaceutical administration. “Pharmaceuticallyacceptable salts” means salts of compounds of the present disclosurewhich are pharmaceutically acceptable, and which possess the desiredpharmacological activity. Such salts include acid addition salts formedwith inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like; or withorganic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonicacid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid,4,4′-methylenebis (3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- anddicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids,benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid,cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonicacid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid,glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid,lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid,mandelic acid, methanesulfonic acid, muconic acid,o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonicacid, phenyl-substituted alkanoic acids, propionic acid,p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid,succinic acid, tartaric acid, tertiarybutylacetic acid, trimethylaceticacid, and the like. Pharmaceutically acceptable salts also include baseaddition salts which may be formed when acidic protons present arecapable of reacting with inorganic or organic bases. Acceptableinorganic bases include sodium hydroxide, sodium carbonate, potassiumhydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organicbases include ethanolamine, diethanolamine, triethanolamine,tromethamine, N-methylglucamine and the like. It should be recognizedthat the particular anion or cation forming a part of any salt of thisinvention is not critical, so long as the salt, as a whole, ispharmacologically acceptable. Additional examples of pharmaceuticallyacceptable salts and their methods of preparation and use are presentedin Handbook of Pharmaceutical Salts Properties, and Use (P. H. Stahl &C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002), which isincorporated herein by reference.

The compounds of the disclosure may contain one or more chiral centersand, therefore, exist as stereoisomers. The term “stereoisomers” whenused herein consist of all enantiomers or diastereomers. These compoundsmay be designated by the symbols “(+),” “(−),” “R” or “S,” depending onthe configuration of substituents around the stereogenic carbon atom,but the skilled artisan will recognize that a structure may denote achiral center implicitly. The present invention encompasses variousstereoisomers of these compounds and mixtures thereof. Mixtures ofenantiomers or diastereomers may be designated “(±)” in nomenclature,but the skilled artisan will recognize that a structure may denote achiral center implicitly.

The compounds of the disclosure may contain one or more double bondsand, therefore, exist as geometric isomers resulting from thearrangement of substituents around a carbon-carbon double bond. Thesymbol

denotes a bond that may be a single, double or triple bond as describedherein. Substituents around a carbon-carbon double bond are designatedas being in the “Z” or “E” configuration wherein the terms “Z” and “E”are used in accordance with IUPAC standards. Unless otherwise specified,structures depicting double bonds encompass both the “E” and “Z”isomers. Substituents around a carbon-carbon double bond alternativelycan be referred to as “cis” or “trans,” where “cis” representssubstituents on the same side of the double bond and “trans” representssubstituents on opposite sides of the double bond.

Compounds of the disclosure may contain a carbocyclic or heterocyclicring and therefore, exist as geometric isomers resulting from thearrangement of substituents around the ring. The arrangement ofsubstituents around a carbocyclic or heterocyclic ring are designated asbeing in the “Z” or “E” configuration wherein the terms “Z” and “E” areused in accordance with IUPAC standards. Unless otherwise specified,structures depicting carbocyclic or heterocyclic rings encompass both“Z” and “E” isomers. Substituents around a carbocyclic or heterocyclicring may also be referred to as “cis” or “trans”, where the term “cis”represents substituents on the same side of the plane of the ring andthe term “trans” represents substituents on opposite sides of the planeof the ring. Mixtures of compounds wherein the substituents are disposedon both the same and opposite sides of plane of the ring are designated“cis/trans.”

Individual enantiomers and diasteriomers of compounds of the presentinvention can be prepared synthetically from commercially availablestarting materials that contain asymmetric or stereogenic centers, or bypreparation of racemic mixtures followed by resolution methods wellknown to those of ordinary skill in the art. These methods of resolutionare exemplified by (1) attachment of a mixture of enantiomers to achiral auxiliary, separation of the resulting mixture of diastereomersby recrystallization or chromatography and liberation of the opticallypure product from the auxiliary, (2) salt formation employing anoptically active resolving agent, (3) direct separation of the mixtureof optical enantiomers on chiral liquid chromatographic columns or (4)kinetic resolution using stereoselective chemical or enzymatic reagents.Racemic mixtures can also be resolved into their component enantiomersby well-known methods, such as chiral-phase liquid chromatography orcrystallizing the compound in a chiral solvent. Stereoselectivesyntheses, a chemical or enzymatic reaction in which a single reactantforms an unequal mixture of stereoisomers during the creation of a newstereocenter or during the transformation of a pre-existing one, arewell known in the art. Stereoselective syntheses encompass both enantio-and diastereoselective transformations, and may involve the use ofchiral auxiliaries. For examples, see Carreira and Kvaerno, Classics inStereoselective Synthesis, Wiley-VCH: Weinheim, 2009.

The compounds disclosed herein can exist in solvated as well asunsolvated forms with pharmaceutically acceptable solvents such aswater, ethanol, and the like, and it is intended that the inventionembrace both solvated and unsolvated forms. In one embodiment, thecompound is amorphous. In one embodiment, the compound is a singlepolymorph. In another embodiment, the compound is a mixture ofpolymorphs. In another embodiment, the compound is in a crystallineform.

The invention also embraces isotopically labeled compounds of theinvention which are identical to those recited herein, except that oneor more atoms are replaced by an atom having an atomic mass or massnumber different from the atomic mass or mass number usually found innature. Examples of isotopes that can be incorporated into compounds ofthe invention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorus, sulfur, fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C,¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶Cl, respectively. For example,a compound of the invention may have one or more H atom replaced withdeuterium.

Certain isotopically-labeled disclosed compounds (e.g., those labeledwith ³H and ¹⁴C) are useful in compound and/or substrate tissuedistribution assays. Tritiated (i.e., ³H) and carbon-14 (i.e., ¹⁴C)isotopes are particularly preferred for their ease of preparation anddetectability. Further, substitution with heavier isotopes such asdeuterium (i.e., ²H) may afford certain therapeutic advantages resultingfrom greater metabolic stability (e.g., increased in vivo half-life orreduced dosage requirements) and hence may be preferred in somecircumstances. Isotopically labeled compounds of the invention cangenerally be prepared by following procedures analogous to thosedisclosed in the examples herein by substituting an isotopically labeledreagent for a non-isotopically labeled reagent.

A “patient” or “subject” as described herein, refers to any animal atrisk for, suffering from or diagnosed for an inflammatory condition,including, but not limited to, mammals, primates, and humans. In certainembodiments, the patient may be a non-human mammal such as, for example,a cat, a dog, or a horse. A patient may be an individual diagnosed witha high risk of developing an inflammatory condition, someone who hasbeen diagnosed with an inflammatory condition, someone who previouslysuffered from an inflammatory condition, or an individual evaluated forsymptoms or indications of an inflammatory condition.

“A patient in need”, as used herein, refers to a patient suffering fromany of the symptoms or manifestations of an inflammatory condition,e.g., inflammatory bowel disease, a patient who may suffer from any ofthe symptoms or manifestations of an inflammatory condition, or anypatient who might benefit from a method of the invention for treating aninflammatory condition. A patient in need may include a patient who isdiagnosed with a risk of developing an inflammatory condition such asinflammatory bowel disease, a patient who has suffered from aninflammatory condition, or a patient who has previously been treated forsuch a condition.

The terms “treat”, “treatment”, “treating” and the like are used hereinto generally mean obtaining a desired pharmacological and/orphysiological effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of partially or completely curing a diseaseand/or adverse effect attributed to the disease. The term “treatment” asused herein covers any treatment of a disease in a mammal, particularlya human, and includes: (a) preventing the disease from occurring in asubject which may be predisposed to the disease but has not yet beendiagnosed as having it; (b) inhibiting the disease, i.e. preventing thedisease from increasing in severity or scope; (c) relieving the disease,i.e. causing partial or complete amelioration of the disease; or (d)preventing relapse of the disease, i.e. preventing the disease fromreturning to an active state following previous successful treatment ofsymptoms of the disease or treatment of the disease.

“Effective amount,” as used herein, refers to the amount of an agentthat is sufficient to at least partially treat a condition whenadministered to a patient. The therapeutically effective amount willvary depending on the severity of the condition, the route ofadministration of the component, and the age, weight, etc. of thepatient being treated. Accordingly, an effective amount of a compound ofthe present disclosure is the amount of such a compound necessary totreat one or more conditions or diseases contemplated herein in apatient such that administration of the agent prevents the condition(s)from occurring in a subject, prevents progression of the condition(e.g., prevents the onset or increased severity of symptoms of thecondition), or relieves or completely ameliorates all associatedsymptoms of the condition, i.e. causes regression of the condition. Aneffective amount may also be the amount of such a compound necessary tobring about a desired biological effect, e.g. decreasing INF-γ levels.

Efficacy of treatment may be evaluated by means of evaluation of grosssymptoms associated with the inflammatory condition, analysis of tissuehistology, biochemical assay, imaging methods such as, for example,magnetic resonance imaging, or other known methods. For instance,efficacy of treatment may be evaluated by analyzing gross symptoms ofthe condition such as changes in tissue inflammation, abdominal pain,vomiting, diarrhea, rectal bleeding, cramps, muscle spasms, weight loss,malnutrition, fever, anemia or other aspects of gross pathologyassociated with an inflammatory condition following administration of acompound described herein.

Efficacy of treatment may also be evaluated at the tissue or cellularlevel, for example, by means of obtaining a tissue biopsy (e.g., agastrointestinal tissue biopsy) and evaluating gross tissue or cellmorphology or staining properties. Biochemical assays that examineprotein or RNA expression may also be used to evaluate efficacy oftreatment. For instance, one may evaluate IL-22, IFN-γ, or levels ofanother protein or gene product indicative of one or more inflammatorycondition(s), inflammatory cytokine production, or an IL-22 mediatedinflammatory response in dissociated cells or non-dissociated tissue viaimmunocytochemical, immunohistochemical, Western blotting, or Northernblotting methods, or methods useful for evaluating RNA levels such asquantitative or semi-quantitative polymerase chain reaction. One mayalso evaluate the presence or level of expression of useful biomarkersfound in fecal matter, plasma, or serum to evaluate disease state andefficacy of treatment.

Methods

The present disclosure provides a method of treating an inflammatorycondition in a patient in need thereof, comprising administering aneffective amount of a compound disclosed herein to the patient.Exemplary inflammatory conditions contemplated herein include Crohn'sdisease, gastroduodenal Crohn's disease, Crohn's (granulomatous)colitis, ulcerative colitis, collagenous colitis, lymphocytic colitis,ischaemic colitis, diversion colitis, Behçet's disease, microscopiccolitis, ulcerative proctitis, proctosigmoiditis, jejunoileitis,left-sided colitis, pancolitis, ileocolitis, ileitis, indeterminatecolitis and eosinophilic enteritis. Other contemplated inflammatoryconditions include cartilage inflammation, bone degradation, rheumatoidarthritis, arthritis, psoriatic arthritis, hypersensitivity pneumonitis,liver diseases such as fatty liver, hepatitis, hepatic steatosis, andnonalcoholic steatohepatitis (NASH), fibrosis (e.g., intestinalfibrosis, lung fibrosis, or liver fibrosis), autoimmune polyendocrinesyndrome, Addison's disease, Goodpasture's syndrome, Graves' disease,Guillain-Barré syndrome, Hashimoto's encephalopathy, psoriasis,Hashimoto's thyroiditis, juvenile arthritis (e.g., juvenile idiopathicarthritis), lupus erythematous, multiple sclerosis, Alzheimer's disease,dermatitis, atopic dermatitis, acne, Type I diabetes mellitus,autoimmune hepatitis, Ménière's disease, Raynaud's phenomenon, Sjögren'ssyndrome, ankylosing spondylitis, chronic fatigue syndrome, rheumaticpolymyalgia, osteoarthritis, prostatitis, and Reiter syndrome. Incertain embodiments the patient is a mammal. In certain otherembodiments the patient is a human.

In certain embodiments, a method of treating an inflammatory disease orcondition is provided, wherein the disease or condition is selected fromthe group consisting of inflammatory bowel disease, cartilageinflammation, bone degradation, ulcerative colitis, psoriasis,arthritis, psoriatic arthritis, rheumatoid arthritis, juvenilearthritis, juvenile rheumatoid arthritis, autoimmune hepatitis, Crohn'sdisease, lupus erythematosus, multiple sclerosis, Alzheimer's disease,dermatitis, atopic dermatitis, acne, Type I diabetes mellitus, Raynaud'sphenomenon, Graves' disease, and Addison's disease, and wherein themethod comprises administering to a patient in need thereof an effectiveamount of a disclosed compound. In certain embodiments the patient is amammal. In certain other embodiments the patient is a human.

A method of treating Crohn's disease is provided, and/or ulcerativecolitis, and/or inflammatory bowel disease, comprising administering toa patient in need thereof an effective amount of a disclosed compound.In certain embodiments the patient is a mammal. In certain otherembodiments the patient is a human.

In certain embodiments, the inflammatory disease or condition isselected from the group consisting of Crohn's disease, ulcerativecolitis, collagenous colitis, lymphocytic colitis, diversion colitis,Behçet's disease, idiopathic inflammatory bowel disease, irritable bowelsyndrome, regional enteritis, spastic colon, microscopic colitis,Crohn's colitis, perianal disease, and indeterminate colitis.

In certain embodiments, the inflammatory disease or condition is Crohn'sdisease. In certain embodiments, the Crohn's disease is selected fromthe group consisting of ileocolitis, ileitis, gastroduodenal Crohn'sdisease, jejunoileitis, and granulomatous ileocolitis. In certainembodiments, the Crohn's disease includes intestinal fibrosis. Incertain embodiments, the Crohn's disease is fibrostenotic Crohn'sdisease.

In another aspect, the present disclosure provides a method ofpreventing, treating, or reducing fibrostenosis or intestinal fibrosisin a subject, comprising administering to a subject in need thereof atherapeutically effective amount of a compound disclosed herein. Incertain embodiments, the fibrostenosis or intestinal fibrosis isassociated with Crohn's disease.

It may be appreciated that inflammatory bowel disease may be associatedwith a number of symptoms. Accordingly, the present disclosure providesa method of relieving one or more symptoms of inflammatory bowel diseaseselected from the group consisting of abdominal pain, vomiting,diarrhea, rectal bleeding, severe cramps, muscle spasms, weight loss,malnutrition, fever, anemia, skin lesions, joint pain, eye inflammation,liver disorders, arthritis, pyoderma gangrenosum, primary sclerosingcholangitis, non-thyroidal illness syndrome, and growth defects inchildren, comprising administering to a patient in need thereof aneffective amount of a disclosed compound.

In one aspect, the present disclosure relates to a method of decreasingIFN-γ levels and/or inhibiting IFN-γ, comprising administering to asubject in need thereof a therapeutically effective amount of adisclosed compound. “Inhibiting IFN-γ,” as used herein, may refer to acomplete or partial reduction in IFN-γ expression or activity. Thus,inhibiting IFN-γ may refer to alterations in IFN-γ gene or chromatinstate or altered interaction with regulators of gene transcription orgene accessibility that results in a complete or partial reduction inexpression of IFN-γ gene products, e.g., IFN-γ RNA, IFN-γ protein, orpeptide sequences of IFN-γ. Inhibiting IFN-γ may also refer toinhibition of processes crucial to IFN-γ gene product expression,including, but not limited to IFN-γ transcription, IFN-γ RNA processing,IFN-γ protein translation, or IFN-γ post-translational modification.Additionally, inhibiting IFN-γ may refer to inhibiting activity of IFN-γgene products, including peptides of IFN-γ, nucleotide products of IFN-γ(e.g., IFN-γ mRNA), and IFN-γ protein. Inhibiting activity of IFN-γ geneproducts may include a reduction in IFN-γ signaling or direct orindirect interaction of IFN-γ with other cellular components (e.g.,proteins, peptides, DNA, RNA, lipids, or signaling molecules) includingnuclear, organelle, cytosolic, membrane, and extracellular components.For example, inhibiting IFN-γ activity may include inhibiting IFN-γbinding or activation of CSF1R or inhibiting CSF1R downstream signalingeffects (e.g., MAP Kinase phosphorylation or macrophage proliferation).

In one aspect, the present disclosure relates to a method of increasingIL-22 levels, comprising administering to a subject in need thereof atherapeutically effective amount of a disclosed compound. Such increasemay be for improving or strengthening immunity or to provide animmunoprotective function.

The invention also provides methods of increasing IL-22 production incells of a patient suffering from an inflammatory condition, comprisingadministering to a patient in need thereof an effective amount of adisclosed compound. IL-22 production may be increased in any cell inwhich IL-22 expression or activity occurs, including cells of thegastrointestinal tract, immune system, and blood. Cells of thegastrointestinal tract (including cells of the stomach, duodenum,jejunum, ileum, colon, rectum and anal canal), include columnarepithelial cells, mucosal epithelial cells, zymogenic cells, neck mucuscells, parietal cells, gastrin cells, goblet cells, paneth cells,oligomucus cells, and villus absorptive cells. Cells of the immunesystem include leukocytes, phagocytes (e.g., macrophages, neutrophils,and dendritic cells), monocytes, mast cells, eosinophils, basophils,natural killer cells, innate cells, lymphocytes, B cells, and T cells.Blood cells include red blood cells (erythrocytes) and white blood cells(leukocytes, monocytes, and platelets).

In one aspect, the present disclosure relates to a method of increasingIL-22 levels and decreasing IFN-γ levels, comprising administering to asubject in need thereof a therapeutically effective amount of a compounddescribed above.

In one aspect, the present disclosure relates to a method of inhibitinglipid peroxidation, comprising administering to a subject in needthereof a therapeutically effective amount of a compound describedabove.

In one aspect, the present disclosure relates to a method of modulatingan aryl hydrocarbon receptor (AhR), comprising administering a selectiveAhR modulator; wherein said selective AhR modulator is a compounddescribed above.

In another aspect, the present invention provides a compound asdescribed herein for use as a medicament. In another aspect, the presentinvention provides a compound as described herein for use in a method oftreating an inflammatory disease or condition. The inflammatory diseaseor condition may be as defined above. In yet another aspect, the presentinvention provides a compound as described herein for use in a method oftreating Crohn's disease and/or ulcerative colitis, and/or inflammatorybowel disease. The Crohn's disease may be as defined above. In anotheraspect, the present disclosure provides a compound as described hereinfor use in a method of preventing, treating, or reducing fibrostenosisor intestinal fibrosis. In certain embodiments, the fibrostenosis orintestinal fibrosis is associated with Crohn's disease. In anotherembodiment, the present invention provides a compound as describedherein for use in a method of relieving one or more symptoms ofinflammatory bowel disease. The symptoms may be as defined above. In oneaspect, the present invention provides a compound as described hereinrelates to a method of decreasing IFN-γ levels and/or inhibiting IFN-γin a subject in need thereof. Inhibiting IFN-γ may be as defined above.In a further aspect, the present provides a compound as described hereinfor use in a method of increasing IL-22 levels in a subject. Suchincrease may be for improving or strengthening immunity or to provide animmunoprotective function. The invention also provides a compound asdescribed above for use in a method of increasing IL-22 production incells of a patient suffering from an inflammatory condition. Theinflammatory condition may be as defined above. In another aspect, thepresent invention provides a compound as described herein for use in amethod of increasing IL-22 levels and decreasing IFN-γ levels in asubject. The present invention also provides a compound as describedherein for use in a method of inhibiting lipid peroxidation in asubject. The present invention also provides a compound for use in amethod of modulating an aryl hydrocarbon receptor (AhR) in a subject.

Pharmaceutical Compositions and Routes of Administration

The present invention also provides pharmaceutical compositionscomprising a compound described herein. In another aspect, the inventionprovides a pharmaceutical composition for use in treating aninflammatory condition. The pharmaceutical composition may comprise acompound described herein and a pharmaceutically acceptable carrier. Asused herein the term “pharmaceutical composition” means, for example, amixture containing a specified amount of a therapeutic compound, e.g., atherapeutically effective amount, of a therapeutic compound in apharmaceutically acceptable carrier to be administered to a mammal,e.g., a human, in order to treat an inflammatory condition.

Compounds of the present disclosure can be specially formulated foradministration of the compound to a subject in solid, liquid or gelform, including those adapted for the following: (1) oraladministration, for example, drenches (aqueous or non-aqueous solutionsor suspensions), lozenges, dragees, capsules, pills, tablets (e.g.,those targeted for buccal, sublingual, and systemic absorption),boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscular,intravenous or epidural injection as, for example, a sterile solution orsuspension, or sustained-release formulation; (3) topical application,for example, as a cream, ointment, or a controlled-release patch orspray applied to the skin; (4) intravaginally or intrarectally, forexample, as a pessary, cream or foam; (5) sublingually; (6) ocularly;(7) transdermally; (8) transmucosally; or (9) nasally. Examples ofdosage forms include, but are not limited to: tablets; caplets;capsules, such as hard gelatin capsules and soft elastic gelatincapsules; cachets; troches; lozenges; dispersions; suppositories;ointments; cataplasms (poultices); pastes; powders; dressings; creams;plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers);gels; liquids such as suspensions (e.g., aqueous or non-aqueous liquidsuspensions, oil-in-water emulsions, or water-in-oil liquid emulsions),solutions, and elixirs; and sterile solids (e.g., crystalline oramorphous solids) that can be reconstituted to provide liquid dosageforms.

Pharmaceutical compositions containing a compound described herein canbe presented in a dosage unit form and can be prepared by any suitablemethod. A pharmaceutical composition should be formulated to becompatible with its intended route of administration. Usefulformulations can be prepared by methods well known in the pharmaceuticalart. For example, see Remington's Pharmaceutical Sciences, 18th ed.(Mack Publishing Company, 1990).

Pharmaceutical formulations preferably are sterile. Sterilization can beaccomplished, for example, by filtration through sterile filtrationmembranes. Where the composition is lyophilized, filter sterilizationcan be conducted prior to or following lyophilization andreconstitution.

The pharmaceutical compositions of the invention can be formulated forparenteral administration, e.g., formulated for injection via theintravenous, intramuscular, subcutaneous, intralesional, orintraperitoneal routes. The preparation of an aqueous composition, suchas an aqueous pharmaceutical composition containing a compound describedherein, will be known to those of skill in the art in light of thepresent disclosure. Typically, such compositions can be prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for using to prepare solutions or suspensions upon the additionof a liquid prior to injection can also be prepared; and thepreparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Solutions of active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. In addition, sterile, fixed oils may be employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid can be used in the preparation ofinjectables. The sterile injectable preparation may also be a sterileinjectable solution, suspension, or emulsion in a nontoxic parenterallyacceptable diluent or solvent, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. In one embodiment, the compound may be suspended in acarrier fluid comprising 1% (w/v) sodium carboxymethylcellulose and 0.1%(v/v) TWEEN™ 80. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents.Generally, dispersions are prepared by incorporating the varioussterilized active ingredients into a sterile vehicle which contains thebasic dispersion medium and the required other ingredients from thoseenumerated above. Sterile injectable solutions of the invention may beprepared by incorporating a compound described herein in the requiredamount of the appropriate solvent with various of the other ingredientsenumerated above, as required, followed by filtered sterilization. Inthe case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum-drying andfreeze-drying techniques which yield a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof. The injectable formulations can besterilized, for example, by filtration through a bacteria-retainingfilter.

The preparation of more, or highly concentrated solutions forintramuscular injection is also contemplated. In this regard, the use ofDMSO as solvent is preferred as this will result in extremely rapidpenetration, delivering high concentrations of the compound to a smallarea.

Suitable preservatives for use in such a solution include benzalkoniumchloride, benzethonium chloride, chlorobutanol, thimerosal and the like.Suitable buffers include boric acid, sodium and potassium bicarbonate,sodium and potassium borates, sodium and potassium 10 carbonate, sodiumacetate, sodium biphosphate and the like, in amounts sufficient tomaintain the pH at between about pH 6 and pH 8, and preferably, betweenabout pH 7 and pH 7.5. Suitable tonicity agents are dextran 40, dextran70, dextrose, glycerin, potassium chloride, propylene glycol, sodiumchloride, and the like, such that the sodium chloride equivalent of thesolution is in the range 0.9 plus or minus 0.2%. Suitable antioxidantsand stabilizers include sodium bisulfite, sodium metabisulfite, sodiumthiosulfite, thiourea and the like. Suitable wetting and clarifyingagents include polysorbate 80, polysorbate 20, poloxamer 282 andtyloxapol. Suitable viscosity-increasing agents include dextran 40,dextran 70, gelatin, glycerin, hydroxyethylcellulose,hydroxymethylpropylcellulose, lanolin, methylcellulose, petrolatum,polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone,carboxymethylcellulose and the like.

In some embodiments, contemplated herein are compositions suitable fororal delivery of a compound described herein, e.g., tablets, thatinclude an enteric coating, e.g., a gastro-resistant coating, such thatthe compositions may deliver the compound to, e.g., the gastrointestinaltract of a patient.

For example, a tablet for oral administration is provided that comprisesgranules (e.g., is at least partially formed from granules) that includea compound described herein, and one or more pharmaceutically acceptableexcipients. Such a tablet may be coated with an enteric coating.Contemplated tablets may include pharmaceutically acceptable excipientssuch as fillers, binders, disintegrants, and/or lubricants, as well ascoloring agents, release agents, coating agents, sweetening, flavoringagents such as wintergreen, orange, xylitol, sorbitol, fructose, andmaltodextrin, and perfuming agents, preservatives and/or antioxidants.

In some embodiments, contemplated pharmaceutical formulations include anintra-granular phase that includes a compound described herein, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable filler. For example, the compound and a filler may be blendedtogether, optionally, with other excipients, and formed into granules.In some embodiments, the intragranular phase may be formed using wetgranulation, e.g. a liquid (e.g., water) is added to the blendedcompound and filler, and then the combination is dried, milled and/orsieved to produce granules. One of skill in the art would understandthat other processes may be used to achieve an intragranular phase.

In some embodiments, contemplated formulations include an extra-granularphase, which may include one or more pharmaceutically acceptableexcipients, and which may be blended with the intragranular phase toform a disclosed formulation.

A disclosed formulation may include an intragranular phase that includesa filler. Exemplary fillers include, but are not limited to, cellulose,gelatin, calcium phosphate, lactose, sucrose, glucose, mannitol,sorbitol, microcrystalline cellulose, pectin, polyacrylates, dextrose,cellulose acetate, hydroxypropylmethyl cellulose, partiallypregelatinized starch, calcium carbonate, and others includingcombinations thereof.

In some embodiments, a disclosed formulation may include a intragranularphase and/or a extragranular phase that includes a binder, which maygenerally function to hold the ingredients of the pharmaceuticalformulation together. Exemplary binders of the invention may include,but are not limited to, the following: starches, sugars, cellulose ormodified cellulose such as hydroxypropyl cellulose, lactose,pregelatinized maize starch, polyvinyl pyrrolidone, hydroxypropylcellulose, hydroxypropylmethyl cellulose, low substituted hydroxypropylcellulose, sodium carboxymethyl cellulose, methyl cellulose, ethylcellulose, sugar alcohols and others including combinations thereof.

Contemplated formulations, e.g., that include an intragranular phaseand/or an extragranular phase, may include a disintegrant such as butare not limited to, starch, cellulose, crosslinked polyvinylpyrrolidone, sodium starch glycolate, sodium carboxymethyl cellulose,alginates, corn starch, crosmellose sodium, crosslinked carboxymethylcellulose, low substituted hydroxypropyl cellulose, acacia, and othersincluding combinations thereof. For example, an intragranular phaseand/or an extragranular phase may include a disintegrant.

In some embodiments, a contemplated formulation includes anintra-granular phase comprising a compound described herein andexcipients chosen from: mannitol, microcrystalline cellulose,hydroxypropylmethyl cellulose, and sodium starch glycolate orcombinations thereof, and an extra-granular phase comprising one or moreof: microcrystalline cellulose, sodium starch glycolate, and magnesiumstearate or mixtures thereof.

In some embodiments, a contemplated formulation may include a lubricant,e.g. an extra-granular phase may contain a lubricant. Lubricants includebut are not limited to talc, silica, fats, stearin, magnesium stearate,calcium phosphate, silicone dioxide, calcium silicate, calciumphosphate, colloidal silicon dioxide, metallic stearates, hydrogenatedvegetable oil, corn starch, sodium benzoate, polyethylene glycols,sodium acetate, calcium stearate, sodium lauryl sulfate, sodiumchloride, magnesium lauryl sulfate, talc, and stearic acid.

In some embodiments, the pharmaceutical formulation comprises an entericcoating. Generally, enteric coatings create a barrier for the oralmedication that controls the location at which the drug is absorbedalong the digestive tract. Enteric coatings may include a polymer thatdisintegrates a different rates according to pH. Enteric coatings mayinclude for example, cellulose acetate phthalate, methylacrylate-methacrylic acid copolymers, cellulose acetate succinate,hydroxylpropylmethyl cellulose phthalate, methylmethacrylate-methacrylic acid copolymers, ethylacrylate-methacrylic acidcopolymers, methacrylic acid copolymer type C, polyvinylacetate-phthalate, and cellulose acetate phthalate.

Exemplary enteric coatings include Opadry® AMB, Acryl-EZE®, Eudragit®grades. In some embodiments, an enteric coating may comprise about 5% toabout 10%, about 5% to about 20%, 8 to about 15%, about 8% to about 20%,about 10% to about 20%, or about 12 to about 20%, or about 18% of acontemplated tablet by weight. For example, enteric coatings may includean ethylacrylate-methacrylic acid copolymer.

For example, in a contemplated embodiment, a tablet is provided thatcomprises or consists essentially of about 0.5% to about 70%, e.g. about0.5% to about 10%, or about 1% to about 20%, by weight of a compounddescribed herein or a pharmaceutically acceptable salt thereof. Such atablet may include for example, about 0.5% to about 60% by weight ofmannitol, e.g. about 30% to about 50% by weight mannitol, e.g. about 40%by weight mannitol; and/or about 20% to about 40% by weight ofmicrocrystalline cellulose, or about 10% to about 30% by weight ofmicrocrystalline cellulose. For example, a disclosed tablet may comprisean intragranular phase that includes about 30% to about 60%, e.g. about45% to about 65% by weight, or alternatively, about 5 to about 10% byweight of a compound described herein, about 30% to about 50%, oralternatively, about 5% to about 15% by weight mannitol, about 5% toabout 15% microcrystalline cellulose, about 0% to about 4%, or about 1%to about 7% hydroxypropylmethylcellulose, and about 0% to about 4%, e.g.about 2% to about 4% sodium starch glycolate by weight.

In another contemplated embodiment, a pharmaceutical tablet formulationfor oral administration of a compound described herein comprises anintra-granular phase, wherein the intra-granular phase includes acompound described herein or a pharmaceutically acceptable salt thereof(such as a sodium salt), and a pharmaceutically acceptable filler, andwhich may also include an extra-granular phase, that may include apharmaceutically acceptable excipient such as a disintegrant. Theextra-granular phase may include components chosen from microcrystallinecellulose, magnesium stearate, and mixtures thereof. The pharmaceuticalcomposition may also include an enteric coating of about 12% to 20% byweight of the tablet. For example, a pharmaceutically acceptable tabletfor oral use may comprise about 0.5% to 10% by weight of a compounddescribed herein or a pharmaceutically acceptable salt thereof, about30% to 50% by weight mannitol, about 10% to 30% by weightmicrocrystalline cellulose, and an enteric coating comprising anethylacrylate-methacrylic acid copolymer.

In another example, a pharmaceutically acceptable tablet for oral usemay comprise an intra-granular phase, comprising about 5 to about 10% byweight of a compound described herein or a pharmaceutically acceptablesalt thereof, about 40% by weight mannitol, about 8% by weightmicrocrystalline cellulose, about 5% by weight hydropropylmethylcellulose, and about 2% by weight sodium starch glycolate; anextra-granular phase comprising about 17% by weight microcrystallinecellulose, about 2% by weight sodium starch glycolate, about 0.4% byweight magnesium stearate; and an enteric coating over the tabletcomprising an ethylacrylate-methacrylic acid copolymer.

In some embodiments the pharmaceutical composition may contain anenteric coating comprising about 13% or about 15%, 16%, 17% or 18% byweight, e.g., AcyrlEZE® (see, e.g., PCT Publication No. WO2010/054826,which is hereby incorporated by reference in its entirety).

The rate at which point the coating dissolves and the active ingredientis released is its dissolution rate. In an embodiment, a contemplatedtablet may have a dissolution profile, e.g. when tested in a USP/EP Type2 apparatus (paddle) at 100 rpm and 37° C. in a phosphate buffer with apH of 7.2, of about 50% to about 100% of a compound described hereinreleasing after about 120 minutes to about 240 minutes, for exampleafter 180 minutes. In another embodiment, a contemplated tablet may havea dissolution profile, e.g. when tested in a USP/EP Type 2 apparatus(paddle) at 100 rpm and 37° C. in diluted HCl with a pH of 1.0, wheresubstantially none of the compound is released after 120 minutes. Acontemplated tablet, in another embodiment, may have a dissolutionprofile, e.g. when tested in USP/EP Type 2 apparatus (paddle) at 100 rpmand 37° C. in a phosphate buffer with a pH of 6.6, of about 10% to about30%, or not more than about 50%, of the compound releasing after 30minutes.

In some embodiments, methods provided herein may further includeadministering at least one other agent that is directed to treatment ofconditions, diseases, and disorders disclosed herein. In one embodiment,contemplated other agents may be co-administered (e.g., sequentially orsimultaneously).

Non-limiting examples of such combination therapy include combination ofone or more compounds of the invention with an anti-inflammatory agent,an antibiotic agent, an immunosuppressant, an inmmunomodulator, or ananalgesic agent.

Agents contemplated include immunosuppressive agents includingglucocorticoids, cytostatics, antibodies, agents acting onimmunophilins, interferons, opioids, TNF binding proteins,mycophenolate, and small biological agents. For example, contemplatedimmunosuppressive agents include, but are not limited to: tacrolimus,cyclosporine, pimecrolimus, sirolimus, everolimus, mycophenolic acid,fingolimod, dexamethasone, fludarabine, cyclophosphamide, methotrexate,azathioprine, leflunomide, teriflunomide, anakinra, anti-thymocyteglobulin, anti-lymphocyte globulin, muromonab-CD3, afutuzumab,rituximab, teplizumab, efalizumab, daclizumab, basiliximab, adalimumab,infliximab, certolizumab pegol, natalizumab, and etanercept. Othercontemplated agents include anti-diarrheals, laxatives, ironsupplements, and calcium or vitamin D or B-12 supplements.

Exemplary formulations include dosage forms that comprise or consistessentially of about 35 mg to about 500 mg of a compound describedherein. For example, formulations that include about 35 mg, 40 mg, 50mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg,150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, or 250 mg of a compounddescribed herein are contemplated. In one embodiment, a formulation mayinclude about 40 mg, 80 mg, or 160 mg of a compound described herein. Insome embodiments, a formulation may include at least 100 μg of acompound described herein. For example, formulations may include about0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 15 mg, 20 mg,or 25 mg of a compound described herein. The amount administered willdepend on variables such as the type and extent of disease or indicationto be treated, the overall health and size of the patient, the in vivopotency of the compound, the pharmaceutical formulation, and the routeof administration. The initial dosage can be increased beyond the upperlevel in order to rapidly achieve the desired blood-level or tissuelevel. Alternatively, the initial dosage can be smaller than theoptimum, and the dosage may be progressively increased during the courseof treatment. Dosing frequency can vary, depending on factors such asroute of administration, dosage amount and the disease being treated.Exemplary dosing frequencies are once per day, once per week and onceevery two weeks. In some embodiments, dosing is once per day for 7 days.

EXAMPLES

The invention is further illustrated by the following examples. Theexamples are provided for illustrative purposes only, and are not to beconstrued as limiting the scope or content of the invention in any way.

Example 1: Synthesis of Sample 2

Sample 2 was prepared as shown below in Scheme 1. Compound numbers usedin this Example pertain only to this Example; Sample 2 is referred toelsewhere as compound 2.

Synthesis of2-(2-(triethylsilyl)-5-(trifluoromethyl)-1H-indol-3-yl)ethanol (3)

A mixture of 2-iodo-4-(trifluoromethyl)aniline (1) (5.00 g, 17.4 mmol),4-(triethylsilyl)but-3-yn-1-ol (2) (3.85 g, 1.2 mmol),bis(diphenylphosphino)ferrocene]palladium(II) chloride (0.64 g, 0.87mmol), lithium chloride (0.732 g, 17.4 mmol) and sodium carbonate (3.7g, 34.8 mmol)) in 50 mL of N,N-dimethylformamide (DMF) was stirred at100° C. for 15 h. The reaction mixture was diluted with water andextracted with ethyl acetate. The organic extract was dried over Na₂SO₄and concentrated under reduced pressure. The crude material was purifiedby silica gel chromatography to afford 7.00 g of the title compound asyellow oil, which contained ˜20% of starting material (2). The productwas used in the next step without further purification. ¹H NMR (300 MHz,CDCl₃) δ 8.2 (bs, 1H), 7.9 (s, 1H), 7.45 (s, 2H), 3.85 (q, J=8.0 Hz,2H), 3.15 (t, J=6.4 Hz, 2H), 1.46 (t, J=6.0 Hz, 1H), 1.2 (m, 15H); MS(APCI+) m/z=344 (M+H).

Synthesis of 2-(5-(trifluoromethyl)-1H-indol-3-yl)ethanol (4)

A solution of2-(2-(triethylsilyl)-5-(trifluoromethyl)-1H-indol-3-yl)ethanol (3) (2.00g, 5.83 mmol) in 15 mL of tetrahydrofuran (THF) was addedtetrabutylammonium fluoride (7.0 mL, 1 M in THF) and the reactionmixture was stirred at room temperature (rt) for 72 h. The reactionmixture was diluted with water and extracted with ethyl acetate. Theorganic extract was dried over Na₂SO₄ and concentrated under reducedpressure. The crude material was purified by silica gel chromatographyto afford 1.02 g (75%) of the title compound as pale yellow oil. ¹H NMR(300 MHz, CDCl₃) δ 8.3 (bs, 1H), 7.96 (s, 1H), 7.45 (s, 2H), 7.2 (s,1H), 3.90 (t, J=6.8 Hz, 2H), 3.05 (t, J=6.8 Hz, 2H); MS (APCI+) m/z=230(M+H).

Synthesis of 3-(2-bromoethyl)-5-(trifluoromethyl)-1H-indole (5)

A solution of 2-(5-(trifluoromethyl)-1H-indol-3-yl)ethanol (4) (1.00 g,4.36 mmol) in THF (10 ml) was added to a solution of triphenyl phosphine(2.30 g, 8.72 mmol) and perbromomethane (4.40 g, 13.1 mmol) in THF (10mL) pre-stirred for 1 h. The resulting mixture was stirred at rt for 3h. The reaction mixture was then filtered and concentrated under reducedpressure. The crude material was purified by silica gel chromatographyto afford 0.66 g (51%) of the title compound as yellow oil. ¹H NMR (300MHz, CDCl₃) δ 8.25 (bs, 1H), 7.9 (s, 1H), 7.4 (s, 2H), 7.2 (s, 1H), 3.7(t, J=7.8 Hz, 2H), 3.27 (t, J=7.8 Hz, 2H).

Synthesis of 3-(2-azidoethyl)-5-(trifluoromethyl)-1H-indole (6)

A mixture of 3-(2-bromoethyl)-5-(trifluoromethyl)-1H-indole (5) (0.66 g,2.26 mmol) and sodium azide (0.44 g; 6.8 mmol) in DMF (10 mL) wasstirred at 70° C. for 4 h. The reaction mixture was diluted with waterand extracted with ethyl acetate. The organic layer was successivelywashed with brine, sodium thiosulfate, dried and concentrated underreduced pressure. The crude material was purified by silica gelchromatography to afford 0.61 g (100%) of the title compound as a brownoil. ¹H NMR (300 MHz, CDCl₃) δ 8.15 (bs, 1H), 7.87 (s, 1H), 7.44 (d,J=1.5 Hz, 2H), 7.19 (s, 1H), 3.55-3.61 (t, J=7 Hz, 2H), 3.08 (t, J=7.2Hz, 2H).

Synthesis of 2-(5-(trifluoromethyl)-(1H-indol-3-yl))ethanamine (7)

A mixture of 3-(2-azidoethyl)-5-(trifluoromethyl)-H-indole (6) (0.61 g,2.45 mmol) and triphenyl phosphine (1.93 g, 7.41 mmol) in methanol (10mL) was stirred at 70° C. for 2 h. The reaction mixture was concentratedunder reduced pressure and the crude material was purified by silica gelchromatography to afford 0.44 g (75%) of the title compound as a brownoil. ¹H NMR (300 MHz, CDCl₃) δ 8.4 (bs, 1H), 7.9 (s, 1H), 7.4 (s, 2H),7.25 (s, 1H), 7.13-7.14 (d, J=2.4 Hz, 1H), 3.04 (m, 4H); MS (APCI−)m/z=326 (M−H).

Synthesis of1-(3,4-dimethoxyphenyl)-6-(trifluoromethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole(Sample 2)

A mixture of 2-(5-(trifluoromethyl)-1H-indol-3-yl))ethanamine (7) (0.40g, 1.76 mmol) and 3,4-dimethoxybenzaldehyde (8) (0.322 g, 1.93 mmol) inacetic acid (8 ml) was stirred at 80° C. for 24 h. The reaction mixturewas concentrated under reduced pressure and the crude material waspurified by silica gel chromatography to afford 0.16 g (33%) of thetitle compound as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 7.9 (s, 1H),7.71 (bs, 1H), 7.33 (m, 1H), 6.84 (d, J=3.8 Hz, 2H), 5.12 (s, 1H), 3.88(s, 3H), 3.81 (s, 3H), 3.15 (m, 1H), 2.88 (m, 2H); MS (APCI+) m/z=377(M+H).

Example 2: Synthesis of Sample 4a

Sample 4a was prepared as shown below in Scheme 2. Compound numbers usedin this Example pertain only to this Example; Sample 4a is referred toelsewhere as compound 4a.

Synthesis of 2-iodo-1-methyl-1H-indole (2)

A solution of 2-iodo-1H-indole (1) (1.50 g, 6.17 mmol) in THF (20 mL)was added to a suspension of 60% NaH (0.37 g, 9.25 mmol) at 0° C. andthe resulting solution was stirred for 10 min. Methyl iodide (1.75 g,12.3 mmol) was added dropwise and the reaction mixture was allowed towarm slowly from 0° C. to rt over 1 h. The reaction was quenched withsaturated NH₄Cl solution (15 mL) and extracted with ethyl acetate. Theorganic layer was dried over Na₂SO₄ and concentrated under reducedpressure. The crude material was purified by silica gel chromatographyto afford 1.27 g (80%) of the title compound as a pale yellow oil. ¹HNMR (400 MHz, CDCl₃) δ 7.71 (d, J=7.6 Hz, 1H), 7.29 (d, J=8 Hz, 1H),7.14 (t, J=7.6 Hz, 1H), 7.06 (t, J=7.6 Hz, 1H), 6.78 (s, 1H), 3.74 (s,3H).

Synthesis of 3-(2-iodo-1-methyl-1H-indol-3-yl)propanal (4)

To a stirred solution of acrolein (3) (1.38 g, 24.7 mmol) andN-methylaniline (0.16 g, 1.48 mmol) at 0° C. was added TFA (0.16 g, 1.48mmol) dropwise and the reaction mixture was stirred at 0° C. for 10 min.2-Iodo-1-methyl-1H-indole (2) (1.27 g, 4.94 mmol) in CH₂Cl₂ (4 mL) wasadded and the reaction mixture was slowly stirred from 0° C. to rt for 3h. The reaction mixture was diluted with water and extracted withdichloromethane (DCM). The organic layer was dried over Na₂SO₄ andconcentrated under reduced pressure. The crude material was purified bysilica gel chromatography to afford 920 mg (59%) of the title compoundas light yellow foam. ¹H NMR (400 MHz, CDCl₃) δ 9.85 (s, 2H), 7.51 (d,J=8.0 Hz, 1H), 7.30 (d, J=8.4 Hz, 1H), 7.16 (t, J=9.8 Hz, 1H), 7.08 (t,J=8 Hz, 1H), 3.75 (s, 3H), 3.75 (s, 3H), 3.09 (t, J=7.6 Hz, 2H), 2.76(t, J=8.2 Hz, 2H); MS (ESI+) m/z=314 (M+H).

Synthesis ofN-(3-(2-iodo-1-methyl-1H-indol-3-yl))propyl-3,4,5-trimethoxyaniline (6)

To a solution of 3-(2-iodo-1-methyl-1H-indol-3-yl)propanal (4) (920 mg,2.93 mmol) and 3,4,5-trimethoxyaniline (5) (806 mg, 4.40 mmol) inmethanol (15 mL) was added 1 drop of acetic acid and the reactionmixture was stirred for 10 min. NaCNBH₃ (0.46 g, 7.32 mmol) was addedand stirring continued for 16 h. The reaction mixture was diluted withwater and extracted with DCM. The organic layer was dried over Na₂SO₄and concentrated under reduced pressure. The crude material was purifiedby silica gel chromatography to afford 1.07 g (74%) of the titlecompound as an off-white foam. ¹H NMR (400 MHz, CDCl₃) δ 7.53 (d, J=7.8Hz, 1H), 7.31 (d, J=8.1 Hz, 1H), 7.16 (t, J=9 Hz, 1H), 7.06 (t, J=7.8Hz, 1H), 5.77 (s, 2H), 3.75 (s, 3H), 3.14 (t, J=7.05 Hz, 2H), 2.88 (t,J=7.2 Hz, 2H), 1.26 (m, 2H); MS (ESI+) m/z=481 (M+H).

Synthesis of9-methyl-1-(3,4,5-trimethoxyphenyl)-2,3,4,9-tetrahydro-1H-pyrido[2,3-b]indole(Sample 4a)

To a solution ofN-(3-(2-iodo-1-methyl-1H-indol-3-yl))propyl)-3,4,5-trimethoxyaniline (6)(1.07 g, 2.22 mmol) in toluene (5 mL) was addedtris(dibenzylideneacetone) dipalladium(0) (0.1 g, 0.108 mmol),2,2′-bis(diphenylphosphino)-1-1″-binaphthalene (0.12 g, 0.216 mmol) andt-BuONa (0.21 g, 2.16 mmol) and the reaction mixture was stirred at 100°C. for 2 h. The mixture was concentrated under reduced pressure and thecrude material was purified by silica gel chromatography to afford 280mg (36%) of the title compound as a white solid. ¹H NMR (400 MHz, CDCl₃)δ 7.45 (d, 1H), 7.15 (m, 3H), 6.19 (s, 2H), 3.82 (s, 3H), 3.71 (m, 8H),3.26 (s, 3H), 2.79 (t, J=6.6 Hz, 2H), 1.87 (m, 2H); MS (ESI+) m/z=353(M+H).

Example 3: Synthesis of Sample 13

Sample 13 was prepared as shown below in Scheme 3. Compound numbers usedin this Example pertain only to this Example; Sample 13 is referred toelsewhere as compound 13.

Synthesis of 3-bromothiophene-2-carbaldehyde (3)

To a solution of lithium diisopropylamide (31.6 mmol) in 50 mL of THF at−78° C. was added 3-bromothiophene (3.0 g, 31 mmol) (1). After stirringfor 1 h, formylpiperidine (3.50 g, 31.6 mmol) (2) was added and thereaction was warmed to 0° C. After 12 h, the reaction mixture waspartitioned between DCM and sat. NH₄Cl and the organic layer wasseparated, dried over MgSO₄, filtered, and concentrated under reducedpressure. The crude material was purified by silica gel chromatographyto afford 1.90 g (54%) of the title compound as a yellow oil. ¹H NMR(400 MHz, CDCl₃) δ 9.99 (s, 1H), 7.71 (d, J=5.2 Hz, 1H), 7.15 (d, J=4.8Hz, 1H).

Synthesis of 3-azidothiophene-2-carbaldehyde (4)

A solution of 3-bromothiophene-2-carbaldehyde (3) (1.90 g, 0.99 mmol)and sodium azide (3.23 g, 50 mmol) in 50 mL of1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU) was heated to50° C. and stirred for 36 h. The reaction mixture was poured onto icewater and then extracted with DCM. The organic extract was dried overNa₂SO₄, filtered, and concentrated under reduced pressure. The crudematerial was purified by silica gel chromatography to afford 1.00 g(66%) of the title compound as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ9.93 (s, 1H), 7.72 (d, J=5.2 Hz, 1H), 7.04 (d, J=5.2 Hz, 1H).

Synthesis of(E)-3-(3-azidothiophen-2-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one(6)

To a mixture of 3-azidothiophene-2-carbaldehyde (4) (0.80 g, 5.22 mmol)and 1-(3,4,5-trimethoxyphenyl)ethanone (5) (1.64 g, 7.84 mmol) in MeOH(5 mL) was added a solution of NaOH (0.62 g, 15.66 mmol) in water (2 mL)and the reaction mixture was stirred at rt for 16 h. The reactionmixture was diluted with water and extracted with DCM. The organicextract was dried over Na₂SO₄, filtered and concentrated under reducedpressure. The crude material was purified by silica gel chromatographyto afford 1.10 g (61%) of the title compound as a yellow solid. ¹H NMR(400 MHz, CDCl₃) δ 7.88 (d, J=15.6 Hz, 1H), 7.42 (d, J=5.2 Hz, 1H), 7.21(d, J=15.2 Hz, 3H), 7.00 (d, J=5.2 Hz, 1H), 3.94 (d, J=5.2 Hz, 9H); MS(ESI+) m/z=346 (M+H).

Synthesis of(4H-thieno[3,2-b]pyrrol-5-yl)(3,4,5-trimethoxyphenyl)methanone (7)

A solution of(E)-3-(3-azidothiophen-2-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one(6) (1.20 g, 3.48 mmol) in 10 mL of xylenes was stirred at 150° C. for30 min. The reaction mixture was concentrated under reduced pressure andthe crude material was purified by silica gel chromatography to afford1.00 g (91%) of the title compound as an off-white solid. ¹H NMR (300MHz, CDCl₃) δ 9.48 (bs, 1H), 7.42 (d, J=5.4 Hz, 1H), 7.20 (s, 2H), 7.11(s, 1H), 7.01 (d, J=5.7 Hz, 1H), 3.94 (d, J=1.8 Hz, 9H); MS (ESI+)m/z=318 (M+H).

Synthesis of 5-(3,4,5-trimethoxybenzyl)-4H-thieno[3,2-b]pyrrole (Sample13)

A mixture of(4H-thieno[3,2-b]pyrrol-5-yl)(3,4,5-trimethoxyphenyl)methanone (7) (0.80g; 2.52 mmol) and sodium borohydride (480 mg, 12.7 mmol) in 2-propanol(10 mL) was stirred in a sealed tube at 100° C. for 2 h. The reactionmixture was cooled and diluted with water and extracted with DCM. Theorganic extract was dried over Na₂SO₄ and concentrated. The crudematerial was purified by silica gel chromatography to afford 280 mg(37%) of the title compound as a white solid. ¹H NMR (300 MHz, CDCl₃) δ7.92 (bs, 1H), 6.99 (d, J=5.1 Hz, 1H), 6.86 (d, J=5.1 Hz, 1H), 6.46 (s,2H), 6.26 (s, 1H), 4.02 (s, 2H), 3.83 (t, J=3.75 Hz, 9H); MS (ESI+)m/z=304 (M+H).

Example 4: Synthesis of Sample 15

Sample 15 was prepared as shown below in Scheme 4. Compound numbers usedin this Example pertain only to this Example; Sample 15 is referred toelsewhere as compound 15.

Synthesis of 6-(trifluoromethyl)benzo[d]thiazole (3)

To a solution of 6-(trifluoromethyl)benzothiazol-2-amine (1) (2.00 g,9.17 mmol) in 20 mL of THF was added isoamyl nitrite (3.22 g, 27.5mmol). The mixture was heated to reflux for 30 min, quenched with water,and extracted with ethyl acetate. The organic extract was dried overNa₂SO₄ and concentrated under reduced pressure. The crude material waspurified by silica gel chromatography to afford 860 mg (46%) of thetitle compound as a yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 9.16 (s,1H), 8.26 (m, 2H), 7.78 (d, J=1.2 Hz, 1H).

Synthesis of 2-amino-5-(trifluoromethyl)benzenethiol (4)

A solution of 6-(trifluoromethyl)benzo[d]thiazole (3) (830 mg, 4.08mmol) and hydrazine monohydrate (1.52 g, 30.6 mmol) in ethanol (20 mL)was heated to reflux for 1.5 h. The mixture was added to a solution ofacetic acid (3 mL) in water (100 mL) and extracted with DCM. The organicextract was dried over Na₂SO₄ and concentrated under reduced pressure.The crude material was purified by silica gel chromatography to afford670 mg (84%) of the title compound as a yellow oil. ¹H NMR (400 MHz,CDCl₃) δ 7.62 (d, J=1.2 Hz, 1H), 7.39 (dd, J=8.4, 2.0 Hz, 1H), 6.75 (d,J=8.4 Hz, 1H), 4.49 (s, 2H), 2.95 (s, 1H).

Synthesis of 2-(3,4-dimethoxybenzyl)-6-(trifluoromethyl)benzo[d]thiazole(Sample 15)

2-amino-5-(trifluoromethyl)benzenethiol (4) (400 mg, 2.07 mmol),2-(3,4-dimethoxyphenyl) acetic acid (5) and Lawesson's reagent (0.29 g,0.72 mmol) in a sealed tube was subjected to microwave heating at 190°C. for 5 min. The crude material was purified by silica gelchromatography to afford 320 mg (43%) of the title compound as a whitesolid. ¹H NMR (400 MHz, CDCl₃) δ 8.08 (s, 1H), 8.06 (s, 1H), 7.69 (d,J=7.5 Hz, 1H), 6.91 (m, 3H), 4.40 (s, 1H), 3.87 (d, J=6.0 Hz, 6H); MS(ESI+) m/z=354 (M+H).

Example 5: Synthesis of Sample 17

Sample 17 was prepared as shown below in Scheme 5. Compound numbers usedin this Example pertain only to this Example; Sample 17 is referred toelsewhere as compound 17.

Synthesis of N-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazol-2-amine(Sample 17)

A mixture of 2-chloro-1H-benzo[d]imidazole (1) (0.40 g, 2.63 mmol),potassium dihydrogenphosphate (0.36 g, 2.63 mmol) and3,4,5-trimethoxyaniline (0.48 g, 2.63 mmol) in 20 mL of n-BuOH wasstirred at 90° C. for 16 h. The reaction mixture was diluted with waterand extracted with ethyl acetate. The organic extract was dried overNa₂SO₄ and concentrated under reduced pressure. The crude material waspurified by silica gel chromatography to afford 0.43 g (54%) of thetitle compound as an off-white solid. ¹H NMR (300 MHz, CDCl₃) δ 10.8 (s,1H), 9.29 (s, 1H), 7.28 (bs, 2H), 7.12 (s, 2H), 6.97 (bs, 2H), 3.79 (s,6H), 3.62 (s, 3H); MS (ESI+) m/z=300 (M+H).

Example 6: Synthesis of Sample 24

Sample 24 was prepared as shown below in Scheme 6. Compound numbers usedin this Example pertain only to this Example; Sample 24 is referred toelsewhere as compound 24.

Synthesis of thiophen-2-amine-TFA salt (2)

To a solution of tert-butyl thiophen-2-ylcarbamate (1) (0.50 g, 2.51mmol) in 10 mL of DCM was added 2,2,2-trifluoroacetic acid (1.43 g,12.55 mmol). The mixture was stirred at rt for 2 h. The reaction mixturewas concentrated under reduced pressure to afford ˜0.50 g of the titlecompound, which was directly used for the next step withoutpurification.

Synthesis of 2-(3,4,5-trimethoxyphenyl)acetyl chloride (4)

To an ice-cold solution of 2-(3,4,5-trimethoxyphenyl)acetic acid (3)(0.40 g, 1.77 mmol) in 10 mL of DCM was added oxalyl chloride (0.67 g,5.30 mmol) followed by 1 drop of DMF. The mixture was stirred from 0° C.to rt for 2 h. The reaction mixture was concentrated under reducedpressure to afford ˜0.50 g of the title compound, which was used in thenext step without purification.

Synthesis of N-(thiophen-2-yl)-2-(3,4,5-trimethoxyphenyl) acetamide(Sample 24)

To an ice-cold solution of thiophen-2-amine-TFA salt (2) (˜0.50 g,crude) and diisopropylethylamine (DIPEA) (0.99 g, 7.65 mmol) in 10 mL ofDCM was added a solution of 2-(3,4,5-trimethoxyphenyl)acetyl chloride(4) (˜0.50 g, crude) in 5 mL of DCM and the reaction mixture was allowedto warm from 0° C. to rt over 2 h with stirring. The reaction mixturewas diluted with water and extracted with DCM. The organic extract wasdried over Na₂SO₄ and concentrated under reduced pressure. The crudematerial was purified by silica gel chromatography to afford 0.25 g ofthe title compound as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 7.80 (bs,1H), 6.87 (m, 1H), 6.81 (m, 1H), 6.56 (m, 1H), 6.51 (s, 2H), 3.86 (s,9H), 3.70 (s, 2H); MS (ESI+) m/z=308 (M+H).

Example 7: Design and In Vitro Activity of Selective Ah ReceptorModulators (SAhRM) Exhibiting Anti-Inflammatory Properties

As a starting point for the development of the presently disclosedcompounds, a group of 47 compounds belonging to a series of1-aryl-1,2,3,4-tetrahydro-β-carboline derivatives (FIG. 1 ) wereevaluated in vitro for their ability to increase the production of IL-22and to decrease the production of IFN-γ (FIG. 23 ). FIG. 24 shows invitro results for a number of compounds described above. The compoundsdecrease the production of IFN-7. Notably, in addition to decreasingIFN-γ production, compounds 15 and 17, for example, also stronglyincreases the production of IL-22.

Conformational Analysis

Conformational analysis of all compounds in the series was carried outin order to determine the spatial position of their substituents and tofind among the lowest energy conformers, their putative bioactiveconformations.

Molecular modeling studies were performed using SYBYL software version6.92. Three dimensional models of all compounds were built from astandard fragments library, and their geometry was subsequentlyoptimized using the Tripos force field including the electrostatic termcalculated from Gasteiger and Hückel atomic charges. The method ofPowell available in the Maximin2 procedure was used for energyminimization until the gradient value was smaller than 0.0001 kcal/molA².

For each compound, a conformational search using the random searchprocess as implemented in SYBYL was performed to identify its lowestenergy conformations. Random conformational searching is a technique tolocate energy minima of a molecule. It involves making random torsionchanges to selected bonds, followed by a minimization. The cycle ofrandom changes and minimization is repeated many times. After each cyclethe new conformation is compared against all others found so far to seeif it is unique. For the random search, the main options used are themaximum hits (n=6) which defined the minimum number of times eachconformation must be found to stop searching for new conformations, theRMS threshold (RMS=0.2 Å) which defined the maximum RMS differencebetween two conformations before they are considered different.

The conformations produced by the random conformational search are fullyoptimized and can be used immediately for their geometry reoptimizationwith the semi-empirical MOPAC package version 6.0 using the HamiltonianAM1 (keywords: PRECISE, NOMM, PARASOK), and Coulson partial atomiccharges were calculated using the same method. Table 1 showsconformational spaces for selected compounds.

TABLE 1 Compounds n E_(min) (kcal) E_(max) (kcal) CTL-01-07-L-A03 280.76 83.82 CTL-01-07-L-A04 16 43.15 47.70 CTL-01-07-L-B05 157 26.3461.71 CTL-10-26-L-C08 126 −5.12 30.49 CTL-10-26-L-C09 66 −27.40 −10.60CTL-10-26-L-C10 26 31.33 40.74 CTL-10-26-L-F07 30 29.75 47.35CTL-10-26-L-F09 97 26.64 51.86 CTL-10-26-L-G10 135 −40.40 −3.65CTL-10-26-L-H04 202 −8.80 20.56 CTL-10-26-L-H05 106 26.05 45.80CTL-10-26-L-H06 49 8.01 13.62 CTL-10-29-L-A05 253 −43.79 −7.10 SGA360853 −95.57 −63.71 Leflunomide 29 −124.59 −121.31 n: number ofconformers, E_(min): lowest energy conformer, E_(max): highest energyconformerLeflunomide, but not its active metabolite, A771726, activates the AhR(Scheme 7). SGA 360 is a SAhRM that exhibits anti-inflammatoryproperties in vivo.

Example 8: AhR Binding

Peripheral blood mononuclear cells (PBMC), isolated from 3 healthysubjects, were isolated by density gradient centrifugation (Lymphoprep;Nycomed Pharma) from 5 mL heparinized blood samples. PBMC wereresuspended in RPMI 1640 supplemented with 10% fetal calf serum at aconcentration of 1×106 cells/mL, cultured in 24-well culture plates(Falcon Plastic) and stimulated with medium alone or anti-CD3/anti-CD28(Miltenyi Biotec) antibodies with or without 100, 200, or 400 nM of AhRbinding compounds (02, 04, 13, 15, 17, or 24), dimethyl sulfoxide or6-formylindolo[3,2-b]carbazole (Ficz, 200 nM). Total RNA was extractedfrom cells cultured for 24 hours. For RNA preparation, cells were lysedin 1 mL of guanidium thiocyanate buffer and subjected tophenol/chloroform extraction using TRizol reagent (Invitrogen). Thesample obtained was quantitated by absorbance at 260 nm andcomplementary DNA (cDNA) was synthetized from 1 mg of total RNA. cDNAwas amplified using the following conditions: denaturation for 1 minuteat 95° C.; annealing for 30 seconds at 58° C. for IFN-γ, and 60° C. forβ-actin; and followed by 30 seconds of extension at 72° C. Primersequences were as follows: human IFN-γ, forward (SEQ ID NO. 1)5′-TGGAGACCATCAAGGAAGAC-3′, reverse (SEQ ID NO.2)5′-GCGTTGGACATTCAAGTCAG-3′. IL-22 was evaluated using commerciallyavailable TaqMan probes (Applied Biosystems). β-actin (forward (SEQ IDNO. 3) 5′-AAGATGACCCAGATCATGTTTGAGACC-3′, reverse (SEQ ID NO. 4)5′-AGCCAGTCCAGACGCAGGAT-3′) was used as a housekeeping gene. Geneexpression was calculated using the ΔΔCt algorithm.

Example 9: Aryl Hydrocarbon Receptor-Driven Signals Inhibit CollagenSynthesis in the Gut

Using both in vitro and in vivo models of fibrosis, we have determinedthat AhR is a regulator of collagen synthesis in the gut, as describedbelow.

Materials and Methods Patients and Samples

Mucosal samples were taken from surgical specimens of 10 patients withfibrostenosing CD (median age, 37 years; range: 27-56 years); 7 of these10 patients were receiving corticosteroids, and the remaining patientswere on corticosteroids and azathioprine. Mucosal samples were alsotaken from 3 patients with ulcerative colitis (UC) undergoing colectomyfor a chronic disease unresponsive to medical treatment and 6 UCpatients undergoing endoscopy for recent flare-ups (median age, 38years; range 29-55 years). Four UC patients were receivingcorticosteroids while the remaining were treated with mesalazine. Normalcontrols included samples taken from 4 patients with irritable bowelsyndrome and from macroscopically and microscopically unaffected areasof 6 patients undergoing colectomy for colon cancer (median age, 49years; range 33-68 years).

Isolation and Culture of Intestinal Fibroblasts

All reagents were purchased from Sigma-Aldrich (Milan, Italy) unlessspecified. Intestinal fibroblasts were isolated and phenotypicallycharacterized as described elsewhere. In all experiments, fibroblastswere used between passages 3 and 8. To examine whether AhR regulatescollagen production, fibroblasts isolated from CD patients were starvedovernight and then stimulated with TGF-β1 (TGF-β; 1 ng/mL; Peprotech EC,London, UK) or TNF-α (15 ng/mL; R&D Systems, Abingdon, UK) in thepresence or absence of Ficz (final concentration, 100-400 nM; Alexis,Milan, Italy) or 2-methyl-2H-pyrazole-3-carboxylic acid (CH223191; finalconcentration 10 μM; Calbiochem, Nottingham, England), an AhRantagonist, for 24-48 hours. At the end, cells were used to extract RNAand cell-free supernatants were analyzed for collagen content.

Induction of Colonic Fibrosis

Trinitrobenzene sulfonic acid (TNBS) was dissolved in 45% ethanol andadministered intrarectally to 8-week-old female balb/c mice for 7 weeksas previously described. Ficz (1 μg/mouse) or AhR antagonist (CH223191;10 μg/mouse) was dissolved in phosphate-buffered saline (PBS) and givenintraperitoneally every 48 hours after the fifth week of TNBSadministration. Control mice were given PBS alone. Mice were examined 3times a week for signs of colitis including weight loss and killed atweek 8; afterwards tissues were collected for histology, RNA analysisand collagen analysis. Colonic sections were stained with H&E and withMasson's trichrome to detect connective deposition. Fibrosis was scoredas mild, moderate, or severe as previously reported.

RNA Extraction, Complementary DNA Preparation and Real Time PolymeraseChain Reaction

RNA isolation, reverse transcription of the RNA and real-time PCR werecarried out as previously described. RNA was extracted by using TRIzolreagent according to the manufacturer's instructions (Invitrogen,Carlsbad, Calif.). A constant amount of RNA (1 μg per sample) wasreverse transcribed into complementary DNA, and this was amplified usingthe following conditions: denaturation for 1 minute at 95° C.; annealingfor 30 seconds at 60° C. for human collagen I (Col1A1), human Col3A1,human alpha smooth muscle actin (α-SMA), and (3-actin, at 58° C. forhuman AhR and mouse Col1A2, followed by 30 seconds of extension at 72°C. Primer sequences were as follows: human Col1A1 (SEQ ID NO. 5)5′-GGACACAGAGGTTTCAGTGG-3′, (SEQ ID NO. 5) 3′-GGTGACTTTGGAGACACAGG-5′;Col3A1 (SEQ ID NO. 6) 5′-GGAGAATGTTGTGCAGTTTGC-3′, (SEQ ID NO. 6)3′-CGTTTGACGTGTTGTAAGAGG-5′; human α-SMA (SEQ ID NO. 7)5′-TCTGGAGATGGTGTCACCCA-3′, (SEQ ID NO. 7) 3′-ACCCACTGTGGTAGAGGTCT-5′;human AhR (SEQ ID NO. 8) 5′-GAGCACAAATCAGAGACTGG-3′, (SEQ ID NO. 9)5′-TGGAGGAAGCATAGAAGACC-3′; mouse Col1A2 (SEQ ID NO. 10)5′-ACACAGTGGTATGGATGGAC-3′, (SEQ ID NO. 10) 3′-CAGGTAGGTATGGTGACACA-5′;3-actin ((SEQ ID NO.3) 5′-AAGATGACCCAGATCATGTTTGAGACC-3′, (SEQ ID NO. 4)5′-AGCCAGTCCAGACGCAGGAT-3′) was used as a housekeeping gene. Geneexpression was calculated using the ΔΔCt algorithm.

Flow Cytometry

To assess the intracellular expression of AhR and the phosphorylated (p)form of p38, Erk1/2, NF-kB/p65 and Smad2/3, cells were fixed with 1%formaldehyde for 20 minutes and subsequently permeabilized with 0.5%saponin in 1% bovine serum albumin and stained with anti-AhR (1:50,final dilution; Abcam, Cambridge, UK), anti-p-p38 (pT180/pY182)-PE(final dilution 1:50; BD Biosciences, San Jose, Calif.), anti-p-ERK1/2(pT202/pY204; pT184/pY186)-PE (final dilution 1:50; BD Biosciences),anti-p-NF-kB/p65-FITC (1:50 final dilution; eBioscience, San Diego,Calif.), and anti-p-Smad2/3 (1:50 final dilution; Cell Signaling,Danvers, Mass.). Appropriate secondary antibody and isotype-matchedcontrols (BD Biosciences) were included in all experiments. Cells wereanalyzed using a FACS Verseflow cytometer and FACS Suite software (BDBiosciences).

Collagen Assay

Total collagen was measured in fibroblasts-free supernatants and mousetissue samples by Sircol Collagen Assay Kit (Biocolor Ltd, Belfast, UK)in accordance with the manufacturer's instructions.

Statistical Analysis

Differences between groups were compared using the Student's t-test.

Results AhR Activation Negatively Regulates Collagen Production byIntestinal Fibroblasts

AhR RNA transcripts were constitutively expressed in fibroblastsisolated from the gut of patients with CD, patients with UC and normalcontrols with no significant differences among groups (FIG. 25A). Flowcytometry analysis showed that nearly 50% of intestinal fibroblastsexpressed AhR in both IBD and controls (FIG. 25B). To determine whetherAhR activation regulates collagen production, fibroblasts isolated fromFS of CD patients were treated with TGF-β 1 or TNF-α, two known inducersof collagen, in the presence or absence of Ficz for 24 hours. Asexpected, stimulation of fibroblasts with TGF-β1 or TNF-α induced asignificant increase of transcripts for Col1A1, Col3A1, and α-SMA, amarker of fibroblast activation (FIGS. 26A-26B). Treatment offibroblasts with Ficz did not alter the basal RNA expression of Col1A1,Col3A1, and α-SMA but significantly reduced TGF-β1 or TNF-α-driven RNAtranscripts for Col1A1, Col3A1, and α-SMA (FIGS. 26A-26B). To furtherassess the role of AhR in the control of collagen expression, CDfibroblasts were stimulated with TGF-β1 or TNF-α in the presence orabsence of CH223191. CH223191 significantly enhanced Col1A1 RNAtranscripts in unstimulated fibroblasts as well as RNA expression ofCol1A1, Col1A3, and α-SMA in fibroblasts stimulated with TGF-β1 or TNF-α(FIGS. 27A-27B). Analysis of soluble forms of collagen in fibroblastculture supernatants confirmed that, in unstimulated cells, CH223191 butnot Ficz collagen significantly up-regulated collagen secretion (FIGS.28A-28D). Moreover, Ficz dose-dependently inhibited TGF-β1 andTNF-α-induced collagen secretion while CH223191 inhibited such asynthesis (FIGS. 28A-28D). Neither Ficz nor CH223191 changed fibroblastviability or proliferation (data not shown).

AhR Controls Map Kinase Activation in CD Fibroblasts

Activation of p38 and ERK1/2 MAP kinases has been involved in the TGF-β1and TNF-α-driven collagen induction. Therefore, we next investigatedwhether the AhR-mediated control of collagen synthesis was associatedwith changes of this intracellular pathway. To this end, we monitoredp38 and ERK1/2 activation by flow-cytometry using specific antibodies,which recognize the phosphorylated/active forms of these proteins.

In unstimulated conditions, the fractions of cells expressing p-p38 orp-ERK1/2 were not affected by Ficz while being significantly increasedby CH223191 (FIGS. 29A-29B). TGF-β1 and TNF-α significantly increase thepercentages of fibroblasts expressing p-p38 and p-ERK1/2, and thiseffect was either decreased or increased by Ficz or CH223191,respectively (FIGS. 29A-29B). TGF-β1 and TNF-α also enhanced thefractions of cells expressing p-Smad2/3 or NF-kB/p65 respectively, butneither Ficz nor CH223191 changed such percentages (FIGS. 29A-29B).

AhR Controls TNBS-Induced Intestinal Fibrosis in Mice

To translate these data in vivo, we used an experimental model ofintestinal fibrosis induced in Balb/c mice by repeated, rectaladministration of low-doses of TNBS. To determine whether AhR activationinterferes with collagen synthesis and fibrosis development, mice weregiven intra-peritoneally either Ficz or CH223191 after the fifth week ofTNBS administration (FIGS. 30A-30B). This time point was selected on thebasis of previous studies showing that deposition of collagen begins atweek 4 after the first TNBS administration. Extent and severity ofinflammation and fibrosis were assessed in animals sacrificed on week 8.As expected, mice treated with repeated doses of TNBS exhibited minimalintestinal inflammation but marked thickening of the colon wall.Masson's trichrome staining of colonic sections and collagen RNA andprotein analysis using whole colonic samples confirmed the increasedcollagen induction in TNBS-treated mice as compared to controls. Micegiven Ficz exhibited a significant reduction of collagen expressionwhile those receiving CH223191 produced more collagen as compared toTNBS-treated mice (FIGS. 30B-30C).

DISCUSSION

This study investigated the role of AhR in the control of intestinalfibrosis. AhR was constitutively expressed in intestinal fibroblastsisolated from FS of CD patients as well as in intestinal fibroblasts ofUC patients and normal controls. Although treatment of CD fibroblastswith Ficz did not modify the basal expression of collagen, inhibition ofAhR with CH223191 led to increased collagen production, suggesting thatconstitutive AhR activation in these cells types is essential to keepcollagen synthesis in check. Fibroblasts isolated from sites of FS in CDhave enhanced capacity to respond to pro-fibrotic cytokines by producingcollagen. Since studies in other systems have shown that AhR negativelyregulates intracellular pathways activated by pro-fibrotic cytokines, itwas next evaluated whether AhR activation is involved in TGF-β and TNF-αinduced collagen production. CD fibroblasts displayed a differentcapacity to synthesize collagen when treated with Ficz or CH223191. Inparticular, Ficz dose-dependently reduced collagen RNA and proteinexpression while inhibition of AhR was followed by enhanced collagenproduction in response to TGF-β and TNF-α. Interestingly however, evenat the greatest doses used in our system, Ficz did not completelyabolish cytokine-induced collagen synthesis, raising the possibilitythat AhR does not control all the cytokine-driven intracellular pathwaysthat lead to collagen production. Indeed, analysis of such signalsrevealed that Ficz abrogated activation of both p38 and ERK1/2 withoutaffecting activation of Smad2/3 and NF-kB in fibroblasts stimulated withTGF-β or TNF-α, respectively. The fact that Ficz-mediated abrogation ofp38 and ERK1/2 activation was accompanied by a 60% reduction ofcytokine-driven collagen synthesis advocates a major role for MAPkinases in the control of collagen production in CD fibroblasts. Thelack of effect of both Ficz and CH223191 on Smad2/3 and NF-kB activationin response to TGF-β or TNF-α is noteworthy, as these findings indicatethat AhR activation does not induce a state of global unresponsivenessin intestinal fibroblasts, perhaps explaining why Ficz or CH223191 didnot affect proliferation and survival of these cells.

The AhR-mediated negative regulation of collagen production wassupported by in vivo studies in mice showing that Ficz was effective forminimizing fibrosis associated with chronic long-term inflammation. Incontrast, mice receiving CH223191 exhibited a more intense collagendeposition as compared to control mice. In these studies, treatment withboth Ficz and CH223191 started at a time point (week 5) that ischaracterized by pathological accumulation of collagen in the colon ofTNBS-treated mice. Therefore, it is unlikely that the AhR-mediatedinhibition of collagen production is secondary to suppression of theongoing colitis.

In conclusion, these results show that AhR activation negativelycontrols collagen synthesis in the gut. These novel findings suggestthat AhR-related compounds could help prevent and/or revert FS inpatients with CD.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles cited herein is incorporated by reference for all purposes.

EQUIVALENTS

The invention can be embodied in other specific forms with departingfrom the essential characteristics thereof. The foregoing embodimentstherefore are to be considered illustrative rather than limiting on theinvention described herein. The scope of the invention is indicated bythe appended claims rather than by the foregoing description, and allchanges that come within the meaning and range of equivalency of theclaims are intended to be embraced therein.

1-61. (canceled)
 62. A compound represented by:

or a pharmaceutically acceptable salt thereof.
 63. A pharmaceuticalcomposition comprising a compound according to claim 62, or apharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier.
 64. A method of treating or reducing fibrostenosisor intestinal fibrosis in a subject, the method comprising administeringto a subject in need thereof a therapeutically effective amount of acompound according to claim 62, or a pharmaceutically acceptable saltthereof.